Cystine knot scaffold platform

ABSTRACT

Provided are non-naturally occurring cystine knot peptides (CKPs) that bind to VEGF-A. Additionally, provided are methods of using non-naturally occurring CKPs that bind to VEGF-A, including diagnostic and therapeutic compositions and methods. Non-naturally CKPs that bind low density lipoprotein receptor-related protein 6 (LRP6) are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. ProvisionalApplication Ser. No. 62/219,063, filed Sep. 15, 2015, which isincorporated herein by reference in its entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 146392026800SEQLIST.txt,date recorded: Sep. 15, 2016, size: 178 KB).

BACKGROUND OF THE INVENTION

The design and engineering of novel proteins from alternative proteinscaffolds has been an emerging field in the last decade with a broadspectrum of applications ranging from structure biology and imagingtools to therapeutic reagents that are currently being tested in theclinic (HK Binz et al., Nat Biotechnol 23, 1257-1268, 2005; HK Binz andA Pluckthun, Curr Opin Biotechnol 16, 459-469, 2005; SS Sidhu and SKoide, Curr Opin Struct Biol 17, 481-487, 2007; A Skerra, Curr OpinBiotechnol 18, 295-304, 2007; C Gronwall and S Stahl, J Biotechnol 140,254-269, 2009; T Wurch et al., Trends Biotechnol 30, 575-582, 2012; SBanta et al., Annu Rev Biomed Eng 15, 93-113, 2013).

Desirable physical properties of potential alternative scaffoldmolecules include high thermal stability and reversibility of thermalfolding and unfolding. Several methods have been applied to increase theapparent thermal stability of proteins and enzymes, including rationaldesign based on comparison to highly similar thermostable sequences,design of stabilizing disulfide bridges, mutations to increase α-helixpropensity, engineering of salt bridges, alteration of the surfacecharge of the protein, directed evolution, and composition of consensussequences (Lehmann and Wyss, Cur Open Biotechnology 12, 371-375, 2001).

Cystine-knot peptides come from a wide range of sources and exhibitdiverse pharmacological activities. They are roughly 30-50 amino acidsin length and contain six conserved cysteine residues which form threedisulfide bonds. One of the disulfides penetrates the macrocycle whichis formed by the two other disulfides and their interconnectingbackbones, thereby yielding a characteristic knotted topology withmultiple loops exposed on the surface. The loops are defined as theamino acid regions which flank the six conserved cysteine residues andare highly variable in nature. Furthermore, the unique arrangement ofthe disulfide bonds renders cystine-knot peptides highly stable tothermal, proteolytic and chemical degradation.

Thus, there is a need to develop small, stable, artificial antibody-likemolecules for a variety of therapeutic and diagnostic applications, suchas ocular diseases and disorders. The present invention meets this andother needs.

BRIEF SUMMARY OF THE INVENTION

In certain embodiments, provided herein is a non-naturally occurringcystine knot peptide (CKP) that binds to vascular endothelial growthfactor A (VEGF-A), wherein the CKP comprises the cystine scaffoldstructure:

Z₁C₁L1C₂L2C₃L3C₄L4C₅L5C₆Z₂

wherein:

Z₁ and Z₂ are any amino acid;

L1 is Loop 1 and has a structure selected from the group consisting of:X₁X₂X₃X₄X₅X₆ (SEQ ID NO: 2), X₁X₂X₃X₄X₅X₆X₇ (SEQ ID NO: 3),X₁X₂X₃X₄X₅X₆X₇X₈ (SEQ ID NO: 4), X₁X₂X₃X₄X₅X₆X₇X₈X₉ (SEQ ID NO: 5), andX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀ (SEQ ID NO: 6), wherein each of X₁-X₁₀ is anyamino acid;

L2 is Loop 2 and has the structure: X₁X₂X₃X₄X₅ (SEQ ID NO: 7), whereineach of X₁-X₅ is any amino acid or an unnatural amino acid;

L3 is Loop 3 and has the structure: X₁X₂X₃, wherein each of X₁-X₃ is anyamino acid or an unnatural amino acid;

L4 is Loop 4 and has the structure: X₁, wherein X₁ is any amino acid oran unnatural amino acid;

L5 is Loop 5 and has the structure: X₁X₂X₃X₄X₅ (SEQ ID NO: 7), whereineach of X₁-X₅ is any amino acid or an unnatural amino acid;

wherein the unnatural amino acid is selected from the group consistingof L-propargylglycine-PEG₆-, L-sulfotyrosine, L-norleucine,L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan,L-3-fluorotyrosine, and L-4-fluorophenylalanine; and wherein the CKPbinds to VEGF-A with an affinity of 500 pM or better.

In certain embodiments according to (or as applied to) any of theembodiments above, the non-naturally occurring (CKP) that binds toVEGF-A has an altered disulfide bond connectivity—with reference to awild-type Ecballium elaterium trypsin inhibitor EETI-II protein havingthe amino acid sequence set forth in SEQ ID NO: 1; wherein the altereddisulfide bond connectivity is C1-C4, C2-C3 and C5-C6.

In certain embodiments according to (or as applied to) any of theembodiments above, the unnatural amino acid is selected from the groupconsisting of L-propargylglycine-PEG₆-, L-sulfotyrosine, L-norleucine,L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan,L-3-fluorotyrosine,-L-4-fluorophenylalanine, gamma-benzyl-L-proline,gamma-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline,4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine,3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine,3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NHmeta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine,3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine,2-naphthyl-L-alanine, L-4,4′-biphenylalanine,3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine,3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine,4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine,benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine(also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine,cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine(also known as L-tert-Leucine), L-cyclopentylglycine,L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine,3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine,2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine,4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine,L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester ofglycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, andglycine morpholine amide.

In certain embodiments according to (or as applied to) any of theembodiments above, Z₁ and/or Z₂ is more than one amino acid, or anunnatural amino acid. In certain embodiments, Z₂ is two amino acids. Incertain embodiments, Z₂ is three amino acids.

In certain embodiments according to (or as applied to) any of theembodiments above, Z₁ and/or Z₂ is G.

In certain embodiments according to (or as applied to) any of theembodiments above, in L1, X₃ is not I; X₅ is not M; and/or X₆ is not R.In certain embodiments according to (or as applied to) any of theembodiments above, in L1: X₁ is an amino acid selected from P, Q, R, T,V, D, N, K, L, and X; X₂ is an amino acid selected from T, D, L, V, I,R, P, N and X; X₃ is an amino acid selected from T, P, M, L, S, F, R,and X; X₄ is an amino acid selected from R, T, Q, D, W, L, E, S, K, andX; X₅ is an amino acid selected from F, P, V, E, K, L, I, and X; X₆ isan amino acid selected from K, N, F, P, L, Y, T, D, M, and X; X₇ is anamino acid selected from Q, W, H and/X; and/or X₈ is an amino acidselected from Y, A, G, D, E, W, S, and X, wherein X is and unnaturalamino acid is selected from the group consisting ofL-propargylglycine-PEG₆-, L-sulfotyrosine, L-norleucine,L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan,L-3-fluorotyrosine, and L-4-fluorophenylalanine. In certain embodimentsaccording to (or as applied to) any of the embodiments above, in L1: X₉is an amino acid selected from L, I, V, D, E and X, wherein X is andunnatural amino acid is selected from the group consisting ofL-propargylglycine-PEG₆-, L-sulfotyrosine, L-norleucine,L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan,L-3-fluorotyrosine, and L-4-fluorophenylalanine. In certain embodimentsaccording to (or as applied to) any of the embodiments above, in L1: X₁₀is an amino acid selected from Y, T, M, N, F, and X, wherein X is andunnatural amino acid is selected from the group consisting ofL-propargylglycine-PEG₆-, L-sulfotyrosine, L-norleucine,L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan,L-3-fluorotyrosine, and L-4-fluorophenylalanine.

In certain embodiments according to (or as applied to) any of theembodiments above, X is and unnatural amino acid is selected from thegroup consisting of L-propargylglycine-PEG₆-, L-sulfotyrosine,L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine,L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine,gamma-benzyl-L-proline, gamma-(4-fluoro-benzyl)-L-proline,4-OH-L-proline, 4-fluoro-L-proline,4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine,3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine,3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NHmeta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine,3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine,2-naphthyl-L-alanine, L-4,4′-biphenylalanine,3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine,3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine,4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine,benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine(also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine,cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine(also known as L-tert-Leucine), L-cyclopentylglycine,L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine,3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine,2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine,4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine,L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester ofglycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, andglycine morpholine amide.

In certain embodiments according to (or as applied to) any of theembodiments above, in L5, each of X₁-X₅ is any amino acid with theexception that X₂ is not proline (P). In certain embodiments accordingto (or as applied to) any of the embodiments above, in L5, each of X₁-X₅is any amino acid with the exception that X₄ is not glycine (G). Incertain embodiments according to (or as applied to) any of theembodiments above, in L5: X₁ is an amino acid selected from G, Q, H, R,L, and Q; X₂ is an amino acid selected from P, M, W, Y, F, L, and H; X₃is an amino acid selected from N, F, H, and Y; X₄ is an amino acidselected from G, Q, D, N, K, H, E, and S; and/or X₅ is an amino acidselected from F, S, and T.

In certain embodiments according to (or as applied to) any of theembodiments above, L1 has the structure X₁X₂X₃X₄X₅X₆X₇X₈ (SEQ ID NO: 4),wherein: X₁ is an amino acid selected from P, Q, and R; X₂ is an aminoacid selected from T, L, and D; X₃ is an amino acid selected from T, Mand L; X₄ is an amino acid selected from R, Q, and D; X₅ is an aminoacid selected from F, P, and V; X₆ is an amino acid selected from K andF; X₇ is an amino acid selected from Q and W; and X₈ is an amino acidselected from Y, G, and D. In certain embodiments according to (or asapplied to) any of the embodiments above, L1 has the structureX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀ (SEQ ID NO: 6), wherein: X₁ is an amino acidselected from Q, R, T and V; X₂ is an amino acid selected from T and D;X₃ is P; X₄ is an amino acid selected from T and W; X₅ is an amino acidselected from F, E, P, and K; X₆ is an amino acid selected from N and P;X₇ is an amino acid selected from W and H; X₈ is an amino acid selectedfrom A, D, E, and W; X₉ is an amino acid selected from L and I; and X₁₀is an amino acid selected from Y, T, M and N.

In certain embodiments according to (or as applied to) any of theembodiments above, in L5: X₁ is an amino acid selected from G, H, and Q;X₂ is an amino acid selected from P, M, W, and Y; X₃ is an amino acidselected from N and Y; X₄ is an amino acid selected from G, Q, and S;and X₅ is an amino acid selected from F and S.

In certain embodiments according to (or as applied to) any of theembodiments above, L1 has the structure X₁X₂X₃X₄X₅X₆X₇X₈ (SEQ ID NO: 4),wherein: X₁ is an amino acid selected from D, Q, N, and K; X₂ is anamino acid selected from V, I, R, L, and P; X₃ is an amino acid selectedfrom L, S, M, T, and F; X₄ is an amino acid selected from Q, L, and E;X₅ is P; X₆ is an amino acid selected from F, L, and Y; X₇ is W; and X₈is G.

In certain embodiments according to (or as applied to) any of theembodiments above, in L5: X₃ is Y; X₅ is S; and X₁, X₂ and X₄ are eachany amino acid, with the exception that X₁ is not G, X₂ is not P, X₄ isnot G, and/or X₅ is not F. In certain embodiments according to (or asapplied to) any of the embodiments above, in L5: X₁ is an amino acidselected from H, L, R, and Q; X₂ is an amino acid selected from W, F,and Y; X₃ is Y; X₄ is an amino acid selected from Q, N, K, H, and E; andX₅ is S.

In certain embodiments according to (or as applied to) any of theembodiments above, L1 has the structure X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀ (SEQ IDNO: 6), wherein: X₁ is an amino acid selected from K, Q, L, and R; X₂ isan amino acid selected from N and D; X₃ is an amino acid selected from Pand L; X₄ is an amino acid selected from L, T, S and K; X₅ is an aminoacid selected from F, V, I, and L; X₆ is an amino acid selected from Nand D; X₇ is W; X₈ is an amino acid selected from A and S; X₉ is anamino acid selected from L, V, E and D; and X₁₀ is an amino acidselected from Y and F.

In certain embodiments according to (or as applied to) any of theembodiments above, in L5: X₁ is Q; X₂ is an amino acid selected from L,F, M, and H; X₃ is an amino acid selected from F, Y, and H; X₄ is anamino acid selected from D, Q, N, and K; and X₅ is an amino acidselected from S and T.

In certain embodiments according to (or as applied to) any of theembodiments above, in L2, X₁ is K, X₂ is Q, X₃ is D, X₄ is S, and X₅ isD.

In certain embodiments according to (or as applied to) any of theembodiments above, L1 has the structure X₁X₂X₃X₄X₅X₆X₇X₈ (SEQ ID NO: 4),wherein: X₅ is P; X₇ is W; X₈ is G; and wherein X₁, X₂, X₃, X₄ and X₆are each any amino acid, with the exception that X₁ is not P, X₂ is notR, X₃ is not I, and/or X₆ is not R. In certain embodiments according to(or as applied to) any of the embodiments above, L1 has the structureX₁X₂X₃X₄X₅X₆X₇X₈ (SEQ ID NO: 4), wherein: X₁ is an amino acid selectedfrom N and D; X₂ is an amino acid selected from I and V; X₃ is an aminoacid selected from M and L; X₄ is an amino acid selected from L, Q, Dand K; X₅ is P; X₆ is an amino acid selected from F, Y, T, L, and M; X₇is W; and X₈ is G.

In certain embodiments according to (or as applied to) any of theembodiments above, in L5: X₁ is an amino acid selected from Q, H, L, andR; X₂ is an amino acid selected from Y and W; X₃ is Y; X₄ is an aminoacid selected from Q and N; and X₅ is S. In certain embodimentsaccording to (or as applied to) any of the embodiments above, in L5: X₃is Y; X₅ is 5; and X₁, X₂, and X₄ are each any amino acid, with theexception that: X₁ is not G, X₂ is not P, and/or X₄ is not G.

In certain embodiments according to (or as applied to) any of theembodiments above, in L2: X₁ is an amino acid selected from G or E; X₂is an amino acid selected from Q, L, P, R, E, and M; X₃ is an amino acidselected from S, D, and N; X₄ is an amino acid selected from F, Y, L, M,and I; and/or X₅ is an amino acid selected from E, D, Q, L, and S,

In certain embodiments according to (or as applied to) any of theembodiments above, in L3, X₁ is L, X₂ is A, and X₃ is G.

In certain embodiments according to (or as applied to) any of theembodiments above, in L4, X₁ is V or F.

In certain embodiments according to (or as applied to) any of theembodiments above, in L5, each of X₁-X₅ is any amino acid with theexception that X₂ is not proline (P).

In certain embodiments according to (or as applied to) any of theembodiments above, in L5, each of X₁-X₅ is any amino acid with theexception that X₄ is not glycine (G).

In certain embodiments according to (or as applied to) any of theembodiments above, in L5: X₁ is any amino acid except G; X₂ is any aminoacid except P; X₃ is any amino acid except N; X₄ is any amino acidexcept G; and/or X₅ is any amino acid except F.

In certain embodiments according to (or as applied to) any of theembodiments above, L1 has the structure X₁X₂X₃X₄X₅X₆X₇X₈ (SEQ ID NO: 4),wherein X₁ is an amino acid selected from N, D, and X; X₂ is an aminoacid selected from I, V, and X; X₃ is M or X; X₄ is an amino acidselected from L, Q, and X; X₅ is P or X; X₆ is F, Y, or X; X₇ is W or X;and X₈ is G or X, wherein X is an unnatural amino acid selected from thegroup consisting of L-propargylglycine-PEG₆-, L-sulfotyrosine,L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine,L-2-chlorotryptophan, L-3-fluorotyrosine, and L-4-fluorophenylalanine.

In certain embodiments according to (or as applied to) any of theembodiments above, X is and unnatural amino acid is selected from thegroup consisting of L-propargylglycine-PEG₆-, L-sulfotyrosine,L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine,L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine,gamma-benzyl-L-proline, gamma-(4-fluoro-benzyl)-L-proline,4-OH-L-proline, 4-fluoro-L-proline,4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine,3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine,3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NHmeta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine,3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine,2-naphthyl-L-alanine, L-4,4′-biphenylalanine,3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine,3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine,4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine,benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine(also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine,cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine(also known as L-tert-Leucine), L-cyclopentylglycine,L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine,3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine,2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine,4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine,L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester ofglycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, andglycine morpholine amide.

In certain embodiments according to (or as applied to) any of theembodiments above, in L3, each of X₁-X₃ is any amino acid or unnaturalamino acid with the exception that X₁ is not Leucine (L), X₂ is notAlanine (A), and X₃ is not glycine (G), wherein the unnatural amino acidselected from the group consisting of L-propargylglycine-PEG₆-,L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine,L-2-chlorotryptophan, L-3-fluorotyrosine, and L-4-fluorophenylalanine.In certain embodiments according to (or as applied to) any of theembodiments above, in L3: X₁ is an amino acid selected from M, F, L V,and X; X₂ is an amino acid selected from S, N, Q, I, Y, E, V, T, and X;X₃ is an amino acid selected from D, Q, T, N, E, R, and X, wherein X isan unnatural amino acid selected from the group consisting ofL-propargylglycine-PEG₆-, L-sulfotyrosine, L-norleucine,L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan,L-3-fluorotyrosine, and L-4-fluorophenylalanine.

In certain embodiments according to (or as applied to) any of theembodiments above, in L4, X₁ is any amino acid except V or an unnaturalamino acid selected from the group consisting ofL-propargylglycine-PEG₆-, L-sulfotyrosine, L-norleucine,L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan,L-3-fluorotyrosine, and L-4-fluorophenylalanine. In certain embodimentsaccording to (or as applied to) any of the embodiments above, in L4, X₁is I, L, or X, wherein X is an unnatural amino acid selected from thegroup consisting of L-propargylglycine-PEG₆-, L-sulfotyrosine,L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine,L-2-chlorotryptophan, L-3-fluorotyrosine, and L-4-fluorophenylalanine.

In certain embodiments according to (or as applied to) any of theembodiments above, in L5: X₃ is Y or X; X₅ is S or X; and X₁, X₂, and X₄are each any amino acid or X, with the exception that X₁ is not G, X₂ isnot P, and/or X₄ is not G, wherein X is an unnatural amino acid selectedfrom the group consisting of L-propargylglycine-PEG₆-, L-sulfotyrosine,L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine,L-2-chlorotryptophan, L-3-fluorotyrosine, and L-4-fluorophenylalanine.In certain embodiments according to (or as applied to) any of theembodiments above, in L5, each of X₁-X₅ is any amino acid with theexception that X₂ is not proline (P). In certain embodiments accordingto (or as applied to) any of the embodiments above, in L5, each of X₁-X₅is any amino acid with the exception that X₄ is not glycine (G). Incertain embodiments according to (or as applied to) any of theembodiments above, in L5: X₁ is an amino acid selected from Q, H, and X;X₂ is an amino acid selected from Y, W, and X; X₃ is Y or X; X₄ is anamino acid selected from Q, N, or X; X₅ is S or X, wherein X is anunnatural amino acid selected from the group consisting ofL-propargylglycine-PEG₆-, L-sulfotyrosine, L-norleucine,L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan,L-3-fluorotyrosine, and L-4-fluorophenylalanine.

In certain embodiments according to (or as applied to) any of theembodiments above, X is and unnatural amino acid is selected from thegroup consisting of L-propargylglycine-PEG₆-, L-sulfotyrosine,L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine,L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine,gamma-benzyl-L-proline, gamma-(4-fluoro-benzyl)-L-proline,4-OH-L-proline, 4-fluoro-L-proline,4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine,3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine,3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NHmeta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine,3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine,2-naphthyl-L-alanine, L-4,4′-biphenylalanine,3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine,3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine,4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine,benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine(also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine,cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine(also known as L-tert-Leucine), L-cyclopentylglycine,L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine,3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine,2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine,4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine,L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester ofglycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, andglycine morpholine amide.

In certain embodiments according to (or as applied to) any of theembodiments above, in L2: X₁ is G or X; X₂ is R, P, or X; X₃ is D or X;X₄ is F, I, or X; and X₅ is E, D, or X, wherein X is an unnatural aminoacid selected from the group consisting of L-propargylglycine-PEG₆-,L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine,L-2-chlorotryptophan, L-3-fluorotyrosine, and L-4-fluorophenylalanine.

In certain embodiments according to (or as applied to) any of theembodiments above, the non-naturally occurring cystine knot peptide(CKP) that binds to vascular endothelial growth factor A (VEGF-A)comprises the amino acid sequence GCNIMLPFWGCGRDFECLQQCICQYYQSCG (SEQ IDNO: 103). In certain embodiments according to (or as applied to) any ofthe embodiments above, the non-naturally occurring cystine knot peptide(CKP) that binds to vascular endothelial growth factor A (VEGF-A)comprises the amino acid sequence GCNIMLPFWGCGRDFECVERCICQYYQSCG (SEQ IDNO: 104). In certain embodiments according to (or as applied to) any ofthe embodiments above, the non-naturally occurring cystine knot peptide(CKP) that binds to vascular endothelial growth factor A (VEGF-A)comprises the amino acid sequence GCNIMLPFWGCGRDFECMSDCICQYYQSCG (SEQ IDNO: 105). In certain embodiments according to (or as applied to) any ofthe embodiments above, the non-naturally occurring cystine knot peptide(CKP) that binds to vascular endothelial growth factor A (VEGF-A)comprises the amino acid sequence GCNIMLPFWGCGRDFECMNQCICQYYQSCG (SEQ IDNO: 106). In certain embodiments according to (or as applied to) any ofthe embodiments above, the non-naturally occurring cystine knot peptide(CKP) that binds to vascular endothelial growth factor A (VEGF-A)comprises the amino acid sequence GCNIMLPFWGCGRDFECMQTCICQYYQSCG (SEQ IDNO: 107). In certain embodiments according to (or as applied to) any ofthe embodiments above, the non-naturally occurring cystine knot peptide(CKP) that binds to vascular endothelial growth factor A (VEGF-A)comprises the amino acid sequence GCNIMLPFWGCGRDFECVYQCICQYYQSCG (SEQ IDNO: 108). In certain embodiments according to (or as applied to) any ofthe embodiments above, the non-naturally occurring cystine knot peptide(CKP) that binds to vascular endothelial growth factor A (VEGF-A)comprises the amino acid sequence GCNIMLPFWGCGRDFECFINCICQYYQSCG (SEQ IDNO: 109). In certain embodiments according to (or as applied to) any ofthe embodiments above, the non-naturally occurring cystine knot peptide(CKP) that binds to vascular endothelial growth factor A (VEGF-A)comprises the amino acid sequence GCNIMLPFWGCGRDFECVSQCICQYYQSCG (SEQ IDNO: 110). In certain embodiments according to (or as applied to) any ofthe embodiments above, the non-naturally occurring cystine knot peptide(CKP) that binds to vascular endothelial growth factor A (VEGF-A)comprises the amino acid sequence GCNIMLPFWGCGRDFECVTECICQYYQSCG (SEQ IDNO: 111). In certain embodiments according to (or as applied to) any ofthe embodiments above, the non-naturally occurring cystine knot peptide(CKP) that binds to vascular endothelial growth factor A (VEGF-A)comprises the amino acid sequence GCNIMLPFWGCGRDFECFYECICQYYQSCG (SEQ IDNO: 112). In certain embodiments according to (or as applied to) any ofthe embodiments above, the non-naturally occurring cystine knot peptide(CKP) that binds to vascular endothelial growth factor A (VEGF-A)comprises the amino acid sequence GCNIMLPFWGCGRDFECMEQCICQYYQSCG (SEQ IDNO: 113). In certain embodiments according to (or as applied to) any ofthe embodiments above, the non-naturally occurring cystine knot peptide(CKP) that binds to vascular endothelial growth factor A (VEGF-A)comprises the amino acid sequence GCNIMLPFWGCGRDFECVYRCICQYYQSCG (SEQ IDNO: 114). In certain embodiments according to (or as applied to) any ofthe embodiments above, the non-naturally occurring cystine knot peptide(CKP) that binds to vascular endothelial growth factor A (VEGF-A)comprises the amino acid sequence GCDVMQPYWGCGPDIDCFVRCLCHWYNSCG (SEQ IDNO: 139). In certain embodiments according to (or as applied to) any ofthe embodiments above, the non-naturally occurring cystine knot peptide(CKP) that binds to vascular endothelial growth factor A (VEGF-A)comprises the amino acid sequence GCDVMQPYWGCGPDIDCLSNCICHWYNSCG (SEQ IDNO: 140). In certain embodiments according to (or as applied to) any ofthe embodiments above, the non-naturally occurring cystine knot peptide(CKP) that binds to vascular endothelial growth factor A (VEGF-A)comprises the amino acid sequence GCNIMLPYWGCGRDFECMEQCICQYYQSCG (SEQ IDNO: 142). In certain embodiments according to (or as applied to) any ofthe embodiments above, the non-naturally occurring cystine knot peptide(CKP) that binds to vascular endothelial growth factor A (VEGF-A)comprises the amino acid sequence GCNIXLPFWGCGRDFECMSDCICQYYQSCG (SEQ IDNO: 144), wherein X is norleucine (Nle). In certain embodimentsaccording to (or as applied to) any of the embodiments above, thenon-naturally occurring cystine knot peptide (CKP) that binds tovascular endothelial growth factor A (VEGF-A) comprises the amino acidsequence GCNIXLPFWGCGRDFECVSQCICQYYQSCG (SEQ ID NO: 145), wherein X isnorleucine (Nle). In certain embodiments according to (or as applied to)any of the embodiments above, the non-naturally occurring cystine knotpeptide (CKP) that binds to vascular endothelial growth factor A(VEGF-A) comprises the amino acid sequenceGCNIXLPYWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 146), wherein X is norleucine(Nle).

In certain embodiments according to (or as applied to) any of theembodiments above, the non-naturally occurring cystine knot peptide(CKP) that binds to vascular endothelial growth factor A (VEGF-A)comprises the amino acid sequence GCDVXQPYWGCGPDIDCLSNCICHWYNSCG (SEQ IDNO: 224), wherein X is norleucine.

In certain embodiments, provided is a non-naturally occurring cystineknot peptide (CKP) comprising the amino acid selected from the groupconsisting of: GCNIMLPFWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 113),GCNIMLPFWGCGRDFECVYRCICQYYQSCG (SEQ ID NO: 114),GCDVMQPYWGCGPDIDCFVRCLCHWYNSCG (SEQ ID NO: 139),GCDVMQPYWGCGPDIDCLSNCICHWYNSCG (SEQ ID NO: 140),GCNIMLPYWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 142),GCNIXLPFWGCGRDFECMSDCICQYYQSCG (SEQ ID NO: 144), wherein X is norleucine(Nle), GCNIXLPFWGCGRDFECVSQCICQYYQSCG (SEQ ID NO: 145), wherein X isnorleucine (Nle), GCNIXLPYWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 146),wherein X is norleucine (Nle), and GCDVXQPYWGCGPDIDCLSNCICHWYNSCG (SEQID NO: 224), wherein X is norleucine. In certain embodiments accordingto (or as applied to) any of the embodiments above, the CKP comprisesthe amino acid sequence set forth in GCNIMLPFWGCGRDFECMEQCICQYYQSCG (SEQID NO: 113). In certain embodiments according to (or as applied to) anyof the embodiments above, the CKP comprises the amino acid sequence setforth in GCNIMLPFWGCGRDFECVYRCICQYYQSCG (SEQ ID NO: 114). In certainembodiments according to (or as applied to) any of the embodimentsabove, the CKP comprises the amino acid sequence set forth inGCDVMQPYWGCGPDIDCFVRCLCHWYNSCG (SEQ ID NO: 139). In certain embodimentsaccording to (or as applied to) any of the embodiments above, the CKPcomprises the amino acid sequence set forth inGCDVMQPYWGCGPDIDCLSNCICHWYNSCG (SEQ ID NO: 140). In certain embodimentsaccording to (or as applied to) any of the embodiments above, the CKPcomprises the amino acid sequence set forth inGCNIMLPYWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 142). In certain embodimentsaccording to (or as applied to) any of the embodiments above, the CKPcomprises the amino acid sequence set forth inGCNIXLPFWGCGRDFECMSDCICQYYQSCG (SEQ ID NO: 144), wherein X is norleucine(Nle). In certain embodiments according to (or as applied to) any of theembodiments above, the CKP comprises the amino acid sequence set forthin GCNIXLPFWGCGRDFECVSQCICQYYQSCG (SEQ ID NO: 145), wherein X isnorleucine (Nle).

In certain embodiments according to (or as applied to) any of theembodiments above, the CKP comprises the amino acid sequence set forthin GCNIXLPYWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 146), wherein X isnorleucine (Nle). In certain embodiments according to (or as applied to)any of the embodiments above, the CKP comprises the amino acid sequenceset forth in GCDVXQPYWGCGPDIDCLSNCICHWYNSCG (SEQ ID NO: 224), wherein Xis norleucine. In certain embodiments according to (or as applied to)any of the embodiments above, the CKP binds VEGF-A.

In certain embodiments, provided is a non-naturally occurring cystineknot peptide (CKP) comprising the amino acid selected from the groupconsisting of: GCDVX₁QPYWGCGPDI-D/E-CLS-N/K/X₂-CICHWYNSCG (SEQ ID NO:534), GCDVX₁QPYWGCGPDI-N/K/X₂-CLS-D/E-CICHWYNSCG (SEQ ID NO: 535),GCNIX₁LPYWGCGRDF-D/E-CME-N/K/X₂-CICQYYQSCG (SEQ ID NO: 538),GCNIX₁LPYWGCGRDF-N/K/X₂-CME-D/E-CICQYYQSCG (SEQ ID NO: 539),GCNIX₁LPFWGCGRDF-D/E-CVS-N/K/X₂-CICQYYQSCG (SEQ ID NO: 540), andGCNIX₁LPFWGCGRDF-N/K/X₂-CVS-D/E-CICQYYQSCG (SEQ ID NO: 541), wherein X₁is norleucine and X₂ is ornithine. In certain embodiments according to(or as applied to) any of the embodiments above, the CKP comprises theamino acid sequence set forth inGCDVX₁QPYWGCGPDI-D/E-CLS-N/K/X₂-CICHWYNSCG (SEQ ID NO: 534), wherein X₁is norleucine and X₂ is ornithine. In certain embodiments according to(or as applied to) any of the embodiments above, the CKP comprises theamino acid sequence set forth in GCDVXQPYWGCGPDIDCLSKCICHWYNSCG (SEQ IDNO: 536), wherein X is norleucine. In certain embodiments according to(or as applied to) any of the embodiments above, the CKP comprises theamino acid sequence set forth in GCDVX₁QPYWGCGPDIDCLSX₂CICHWYNSCG (SEQID NO: 537), wherein X₁ is norleucine and X₂ is ornithine. In certainembodiments according to (or as applied to) any of the embodimentsabove, the CKP comprises the amino acid sequence set forth inGCDVX₁QPYWGCGPDI-N/K/X₂-CLS-D/E-CICHWYNSCG (SEQ ID NO: 535), wherein X₁is norleucine and X₂ is ornithine. In certain embodiments according to(or as applied to) any of the embodiments above, the CKP comprises theamino acid sequence set forth inGCNIX₁LPYWGCGRDF-D/E-CME-N/K/X₂-CICQYYQSCG (SEQ ID NO: 538), wherein X₁is norleucine and X₂ is ornithine. In certain embodiments according to(or as applied to) any of the embodiments above, the CKP comprises theamino acid sequence set forth in GCNIXLPYWGCGRDFECMEKCICQYYQSCG (SEQ IDNO: 543), wherein X is norleucine. In certain embodiments according to(or as applied to) any of the embodiments above, the CKP comprises theamino acid sequence set forth in GCNIX₁LPYWGCGRDFECMEX₂CICQYYQSCG (SEQID NO: 544), wherein X₁ is norleucine and X₂ is ornithine. In certainembodiments according to (or as applied to) any of the embodimentsabove, the CKP comprises the amino acid sequence set forth inGCNIX₁LPYWGCGRDF-N/K/X₂-CME-D/E-CICQYYQSCG (SEQ ID NO: 539), wherein X₁is norleucine and X₂ is ornithine. In certain embodiments according to(or as applied to) any of the embodiments above, the CKP comprises theamino acid sequence set forth inGCNIX₁LPFWGCGRDF-D/E-CVS-N/K/X₂-CICQYYQSCG (SEQ ID NO: 540), wherein X₁is norleucine and X₂ is ornithine. In certain embodiments according to(or as applied to) any of the embodiments above, the CKP comprises theamino acid sequence set forth in GCNIXLPFWGCGRDFECVSKCICQYYQSCG (SEQ IDNO: 545), wherein X is norleucine. In certain embodiments according to(or as applied to) any of the embodiments above, the CKP comprises theamino acid sequence set forth in GCNIX₁LPFWGCGRDFECVSX₂CICQYYQSCG (SEQID NO: 546), wherein X₁ is norleucine and X₂ is ornithine. In certainembodiments according to (or as applied to) any of the embodimentsabove, the CKP comprises the amino acid sequence set forth inGCNIX₁LPFWGCGRDF-N/K/X₂-CVS-D/E-CICQYYQSCG (SEQ ID NO: 541), wherein X₁is norleucine and X₂ is ornithine. In certain embodiments according to(or as applied to) any of the embodiments above, the CKP binds VEGF-A.

In certain embodiments according to (or as applied to) any of theembodiments above, provided is a non-naturally occurring cystine knotpeptide (CKP) that binds to VEGF-A, wherein the CKP comprises thecystine scaffold structure:

Z₁C₁L1C₂L2C₃L3C₄L4C₅L5C₆Z₂

wherein:

Z₁ and Z₂ are any amino acid;

L1 is Loop 1 and has a structure selected from the group consisting of:X₁X₂X₃X₄X₅X₆X₇X₈, X₁X₂X₃X₄X₅X₆X₇X₈X₉, and X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀, whereineach of X₁-X₁₀ is any amino acid;

L2 is Loop 2 and has the structure: X₁X₂X₃X₄X₅, wherein each of X₁-X₅ isany amino acid;

L3 is Loop 3 and has the structure: X₁X₂X₃ wherein each of X₁-X₃ is anyamino acid;

L4 is Loop 4 and has the structure: X₁, wherein X₁ is any amino acid;

L5 is Loop 5 and has the structure: X₁X₂X₃X₄X₅, wherein each of X₁-X₅ isany amino acid;

wherein the CKP has an altered disulfide bond connectivity withreference to a wild-type Ecballium elaterium trypsin inhibitor EETI-IIprotein having the amino acid sequence set forth in SEQ ID NO: 1;wherein the altered disulfide bond connectivity is C1-C4, C2-C3 andC5-C6; and wherein the CKP has a percent alpha helix content of at least20%.

In certain embodiments according to (or as applied to) any of theembodiments above, Z₁ and Z₂ are any amino acid, more than one aminoacid, or an unnatural amino acid. In certain embodiments according to(or as applied to) any of the embodiments above, each of X₁-X₁₀ in L1 isany amino acid or an unnatural amino acid. In certain embodimentsaccording to (or as applied to) any of the embodiments above, each ofX₁-X₅ in L2 is any amino acid or an unnatural amino acid. In certainembodiments according to (or as applied to) any of the embodimentsabove, each of X₁-X₃ in L3 is any amino acid or an unnatural amino acid.In certain embodiments according to (or as applied to) any of theembodiments above, X₁ in L4 is any amino acid or an unnatural aminoacid. In certain embodiments according to (or as applied to) any of theembodiments above, each of X₁-X₅ in L5 is any amino acid or an unnaturalamino acid. In certain embodiments, the unnatural amino acid is selectedfrom the group consisting of: L-propargylglycine-PEG₆-, L-sulfotyrosine,L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine,L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine,gamma-benzyl-L-proline, gamma-(4-fluoro-benzyl)-L-proline,4-OH-L-proline, 4-fluoro-L-proline,4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine,3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine,3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NHmeta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine,3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine,2-naphthyl-L-alanine, L-4,4′-biphenylalanine,3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine,3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine,4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine,benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine(also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine,cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine(also known as L-tert-Leucine), L-cyclopentylglycine,L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine,3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine,2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine,4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine,L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester ofglycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, andglycine morpholine amide.

In certain embodiments according to (or as applied to) any of theembodiments above, the non-naturally occurring (CKP) that binds toVEGF-A binds to VEGF-A with an affinity of 500 pM or less. In certainembodiments according to (or as applied to) any of the embodimentsabove, the binding affinity is determined via surface plasmon resonance.

In certain embodiments according to (or as applied to) any of theembodiments above, Z₁ and/or Z₂ is more than one amino acid, or anunnatural amino acid. In certain embodiments, Z₂ is two amino acids. Incertain embodiments, Z₂ is three amino acids. In certain embodimentsaccording to (or as applied to) any of the embodiments above, in L5,each of X₁-X₅ is any amino acid with the exception that X₂ is notproline (P). In certain embodiments according to (or as applied to) anyof the embodiments above, in L5, each of X₁-X₅ is any amino acid withthe exception that X₄ is not glycine (G).

In certain embodiments according to (or as applied to) any of theembodiments above, the C-terminal carboxyl group of the non-naturallyoccurring (CKP) that binds to VEGF-A is modified (such as capped). Incertain embodiments according to (or as applied to) any of theembodiments above, the N-terminal amine group of the non-naturallyoccurring (CKP) that binds to VEGF-A is modified (such as capped). Incertain embodiments according to (or as applied to) any of theembodiments above, the C-terminal carboxyl group of the non-naturallyoccurring (CKP) that binds to VEGF-A is capped and the N-terminal aminegroup of the non-naturally occurring (CKP) that binds to VEGF-A ismodified (such as capped).

In certain embodiments according to (or as applied to) any of theembodiments above, the C-terminal carboxyl group of the non-naturallyoccurring (CKP) that binds to VEGF-A is amidated. In certain embodimentsaccording to (or as applied to) any of the embodiments above, theN-terminal amine group of the non-naturally occurring (CKP) that bindsto VEGF-A is acetylated. In certain embodiments according to (or asapplied to) any of the embodiments above, the C-terminal carboxyl groupof the non-naturally occurring (CKP) that binds to VEGF-A is amidatedand the N-terminal amine group of the non-naturally occurring (CKP) thatbinds to VEGF-A is acetylated.

In certain embodiments according to (or as applied to) any of theembodiments above, the non-naturally occurring (CKP) that binds toVEGF-A inhibits VEGF-A activity. In certain embodiments according to (oras applied to) any of the embodiments above, CKP inhibits VEGF-Aactivity with and IC₅₀ between about 0.5 nM and about 1.0 nM. In certainembodiments according to (or as applied to) any of the embodimentsabove, the non-naturally occurring EETI-II scaffold protein binds humanVEGF-A, mouse VEGF-A, and rat VEGF-A.

In certain embodiments according to (or as applied to) any of theembodiments above, the non-naturally occurring CKP competes with theantibody G6.31 for binding to VEGF-A. In certain embodiments accordingto (or as applied to) any of the embodiments above, provided is anon-naturally occurring CKP that competes with the non-naturallyoccurring (CKP) that binds to VEGF-A of any one of embodiments above forbinding to VEGF-A.

In certain embodiments according to (or as applied to) any of theembodiments above, non-naturally occurring CKP that binds to an epitopeon VEGF-A comprising at least one of the amino acid residues selectedfrom the group consisting of: V14, V15, F17, D19, Y21, Q22, Y25, I46,K48, N62, D63, L66, M81, 183, K84, P85, H86, G88, Q89, I91, C104, R105,and P106.

In certain embodiments according to (or as applied to) any of theembodiments above, the residues are selected from the group consistingof: K48, N62, and D63. In certain embodiments according to (or asapplied to) any of the embodiments above, the residues are selected fromthe group consisting of: Y21, Y25, and P106. In certain embodimentsaccording to (or as applied to) any of the embodiments above, theresidues are selected from the group consisting of: H86 and Q89. Incertain embodiments according to (or as applied to) any of theembodiments above, the residues are selected from the group consistingof: M81, D19, and Q22. In certain embodiments according to (or asapplied to) any of the embodiments above, the residues are selected fromthe group consisting of: F17, M81, and I91. In certain embodimentsaccording to (or as applied to) any of the embodiments above, theresidues are selected from the group consisting of: V14, F17, D19, Q22,M81, and I91. In certain embodiments according to (or as applied to) anyof the embodiments above, the residues are selected from the groupconsisting of: Y25.

In certain embodiments according to (or as applied to) any of theembodiments above, the non-naturally occurring CKP that binds to VEGF-Ais conjugated to a therapeutic agent. In certain embodiments accordingto (or as applied to) any of the embodiments above, the non-naturallyoccurring CKP that binds to VEGF-A is conjugated to a label. In certainembodiments according to (or as applied to) any of the embodimentsabove, the label is selected from the group consisting of aradioisotope, a fluorescent dye, and an enzyme.

In certain embodiments according to (or as applied to) any of theembodiments above, provided is an isolated nucleic acid encoding thenon-naturally occurring (CKP) that binds to VEGF-A of any one ofembodiments above. Also provided is an expression vector encoding thenucleic acid molecule of any one of the embodiments above. Also providedis a cell comprising the expression vector of any one of the embodimentsabove. Also provided is a method of producing the non-naturallyoccurring (CKP) that binds to VEGF-A of any one of embodiments above,comprising culturing the cell of any one of the embodiments above, andrecovering the non-naturally occurring (CKP) that binds to VEGF-A fromthe cell culture.

Also provided is a method of producing the non-naturally occurring (CKP)that binds to VEGF-A of any one of embodiments above, comprisingchemically synthesizing the non-naturally occurring (CKP) that binds toVEGF-A.

Provided herein is a composition comprising the non-naturally occurring(CKP) that binds to VEGF-A of any one of the embodiments above and apharmaceutically acceptable carrier. In certain embodiments according to(or as applied to) any of the embodiments above, the compositioncomprises one or more additional compounds. In certain embodimentsaccording to (or as applied to) any of the embodiments above, theadditional compound binds to a second biological molecule selected fromthe group consisting of interleukin-6 (IL-6); interleukin-6 receptor(IL-6R); PDGF; angiopoietin; angiopoietin 2; Tie2; S1P; integrins αvβ3,αvβ5, and α5β1; betacellulin; apelin/APJ; erythropoietin; complementfactor D; TNFα; HtrA1; a VEGF receptor; ST-2 receptor; and proteinsgenetically linked to age-related macular degeneration (AMD) risk suchas complement pathway components C2, factor B, factor H, CFHR3, C3b, C5,C5a, C3a, HtrA1, ARMS2, TIMP3, HLA, interleukin-8 (IL-8), CX3CR1, TLR3,TLR4, CETP, LIPC, COL10A1, and TNFRSF10A. In certain embodimentsaccording to (or as applied to) any of the embodiments above, theadditional compound is a non-naturally occurring CKP. In certainembodiments according to (or as applied to) any of the embodimentsabove, the additional compound is an antibody or antigen-bindingfragment thereof.

Provided herein is a method of treating an ocular disease characterizedby angiogenesis and/or vascular permeability or leakage in a subject,comprising administering an effective amount of the non-naturallyoccurring (CKP) that binds to VEGF-A of any one of embodiments above tothe subject. In certain embodiments according to (or as applied to) anyof the embodiments above, the method further comprises administering oneor more additional compounds. In certain embodiments according to (or asapplied to) any of the embodiments above, the non-naturally occurringCKP that binds to VEGF-A is administered simultaneously with theadditional compound(s). In certain embodiments according to (or asapplied to) any of the embodiments above, the non-naturally occurringCKP that binds to VEGF-A is administered before or after the additionalcompound(s). In certain embodiments according to (or as applied to) anyof the embodiments above, the additional compound binds to a secondbiological molecule selected from the group consisting of interleukin-6(IL-6); interleukin-6 receptor (IL-6R); PDGF; angiopoietin; angiopoietin2; Tie2; S1P; integrins αvβ3, αvβ5, and α5β1; betacellulin; apelin/APJ;erythropoietin; complement factor D; TNFα; HtrA1; a VEGF receptor; ST-2receptor; and proteins genetically linked to age-related maculardegeneration (AMD) risk such as complement pathway components C2, factorB, factor H, CFHR3, C3b, C5, C5a, C3a, HtrA1, ARMS2, TIMP3, HLA,interleukin-8 (IL-8), CX3CR1, TLR3, TLR4, CETP, LIPC, COL10A1, andTNFRSF10A. In certain embodiments according to (or as applied to) any ofthe embodiments above, the additional compound is a non-naturallyoccurring CKP. In certain embodiments according to (or as applied to)any of the embodiments above, the additional compound is an antibody orantigen-binding fragment thereof. In certain embodiments according to(or as applied to) any of the embodiments above, the ocular disease isan intraocular neovascular disease selected from the group consisting ofproliferative retinopathies, choroidal neovascularization (CNV),age-related macular degeneration (AMD), diabetic and otherischemia-related retinopathies, diabetic macular edema, pathologicalmyopia, von Hippel-Lindau disease, histoplasmosis of the eye, retinalvein occlusion (RVO), including Central Retinal Vein Occlusion (CRVO)and branched retinal vein occlusion (BRVO), corneal neovascularization,retinal neovascularization, and retinopathy of prematurity (ROP).

In certain embodiments according to (or as applied to) any of theembodiments above, the non-naturally occurring (CKP) that binds toVEGF-A or the composition is administered to the subject via animplantable device. In certain embodiments according to (or as appliedto) any of the embodiments above, the implantable device selected fromthe group consisting of: an ocular insert, a slow-release depot, anocular plug/reservoir, an non-biodegradable ocular implant or abiodegradable ocular implant.

In certain embodiments according to (or as applied to) any of theembodiments above, provided is a composition comprising thenon-naturally occurring (CKP) that binds to VEGF-A of any one ofembodiments above for use in treating an ocular disease characterized byangiogenesis and/or vascular permeability or leakage in a subject. Incertain embodiments according to (or as applied to) any of theembodiments above, the ocular disease is an intraocular neovasculardisease selected from the group consisting of proliferativeretinopathies, choroidal neovascularization (CNV), age-related maculardegeneration (AMD), diabetic and other ischemia-related retinopathies,diabetic macular edema, pathological myopia, von Hippel-Lindau disease,histoplasmosis of the eye, retinal vein occlusion (RVO), includingCentral Retinal Vein Occlusion (CRVO) and branched retinal veinocclusion (BRVO), corneal neovascularization, retinalneovascularization, and retinopathy of prematurity (ROP). In certainembodiments according to (or as applied to) any of the embodimentsabove, the composition is administered to the subject via an implantabledevice. In certain embodiments according to (or as applied to) any ofthe embodiments above, the implantable device selected from the groupconsisting of: an ocular insert, a slow-release depot, an ocularplug/reservoir, an non-biodegradable ocular implant or a biodegradableocular implant.

In certain embodiments according to (or as applied to) any of theembodiments above, provided is a composition comprising thenon-naturally occurring (CKP) that binds to VEGF-A of any one ofembodiments above for use in treating an ocular disease characterized byangiogenesis and/or vascular permeability or leakage in a subject. Incertain embodiments according to (or as applied to) any of theembodiments above, the ocular disease is an intraocular neovasculardisease selected from the group consisting of proliferativeretinopathies, choroidal neovascularization (CNV), age-related maculardegeneration (AMD), diabetic and other ischemia-related retinopathies,diabetic macular edema, pathological myopia, von Hippel-Lindau disease,histoplasmosis of the eye, retinal vein occlusion (RVO), includingCentral Retinal Vein Occlusion (CRVO) and branched retinal veinocclusion (BRVO), corneal neovascularization, retinalneovascularization, and retinopathy of prematurity (ROP). In certainembodiments according to (or as applied to) any of the embodimentsabove, the medicament is administered to the subject via an implantabledevice. In certain embodiments according to (or as applied to) any ofthe embodiments above, the implantable device selected from the groupconsisting of: an ocular insert, a slow-release depot, an ocularplug/reservoir, an non-biodegradable ocular implant or a biodegradableocular implant.

In certain embodiments according to (or as applied to) any of theembodiments above, the non-naturally occurring (CKP) that binds toVEGF-A is formulated for long acting delivery.

Provided herein is a formulation comprising the non-naturally occurring(CKP) that binds to VEGF-A of any of embodiments above and PLGA. Incertain embodiments according to (or as applied to) any of theembodiments above, the PLGA is a PLGA rod.

Also provided herein is a non-naturally occurring cystine knot peptide(CKP) that binds to human low density lipoprotein receptor-relatedprotein 6 (LRP6), wherein the CKP comprises the cystine scaffoldstructure:

Z₁C₁L1C₂L2C₃L3C₄L4C₅L5C₆Z₂;

wherein:

-   -   Z₁ and Z₂ are any amino acid;    -   L1 is Loop 1 and has a structure selected from the group        consisting of: X₁X₂X₃X₄X₅X₆, X₁X₂X₃X₄X₅X₆X₇, X₁X₂X₃X₄X₅X₆X₇X₈,        X₁X₂X₃X₄X₅X₆X₇X₈X₉, and X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀, wherein each of        X₁-X₁₀ is any amino acid;    -   L2 is Loop 2 and has the structure: X₁X₂X₃X₄X₅, wherein each of        X₁-X₅ is any amino acid;    -   L3 is Loop 3 and has the structure: X₁X₂X₃ wherein each of X₁-X₃        is any amino acid;    -   L4 is Loop 4 and has the structure: X₁, wherein X₁ is any amino        acid; and    -   L5 is Loop 5 and has the structure: X₁X₂X₃X₄X₅, wherein each of        X₁-X₅ is any amino acid.

In certain embodiments according to (or as applied to) any of theembodiments above, Z₁ and/or Z₂ is more than one amino acid, or anunnatural amino acid. In certain embodiments, Z₂ is two amino acids. Incertain embodiments, Z₂ is three amino acids.

In certain embodiments according to (or as applied to) any of theembodiments above, Z₁ and/or Z₂ is G.

In certain embodiments according to (or as applied to) any of theembodiments above, in L1: X₁ is an amino acid selected from R, V, M, A,G, N, S, and E; X₂ is an amino acid selected from T, N, S, G, R, and A;X₃ is an amino acid selected from N, R, H, V, K, S, G, I, and Y; X₄ isan amino acid selected from R, V, N, I, K, S, and T; X₅ is an amino acidselected from V, R, K, I, T, S, L, and N; and X₆ is an amino acidselected from K, G, A, I, R, N, S, and V. In certain embodimentsaccording to (or as applied to) any of the embodiments above, in L1: X₇is an amino acid selected from G, R, K, E, P, and T. In certainembodiments according to (or as applied to) any of the embodimentsabove, in L1: X₈ is an amino acid selected from G, R, K, Q, A, and S. Incertain embodiments according to (or as applied to) any of theembodiments above, in L1: X₉ is an amino acid selected from R or G. Incertain embodiments according to (or as applied to) any of theembodiments above, in L1: X₁₀ is an amino acid selected from E, W, andG.

In certain embodiments according to (or as applied to) any of theembodiments above, in L5: X₁ is an amino acid selected from G, S, N, Y,A, and R; X₂ is an amino acid selected from P, G, S, V, E, R, F, and D;X₃ is an amino acid selected from N, G, S, E, P, K, H, and R; X₄ is anamino acid selected from G, R, H, S, Q, V, and D; and X₅ is an aminoacid selected from F, D, N, R, G, Y, S, and T.

In certain embodiments according to (or as applied to) any of theembodiments above, in L2, X₁ is K, X₂ is Q, X₃ is D, X₄ is S, and X₅ isD. In certain embodiments according to (or as applied to) any of theembodiments above, in L3, X₁ is L, X₂ is A, and X₃ is G. In certainembodiments according to (or as applied to) any of the embodimentsabove, in L4, X₁ is V.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the structure of the EETI-II cystine knot protein.

FIG. 2A shows the results of experiments that were performed todetermine whether EGF_CKP9.54.90 disrupts the interaction betweenVEGF-A(8-109) and KDR; VEGF-A(8-109) and Flt-1; VEGF-A 165 and KDR;VEGF-B and Flt-1; VEGF-C and Flt-4; VEGF-D and Flt-4; and PIGF-2 andFLT-1.

FIG. 2B shows the results of experiments that were performed todetermine whether EGF_CKP9.54.90 disrupts the interaction betweenVEGF-A(8-109) and KDR; VEGF-A(8-109) and Flt-1; VEGF-A 165 and KDR; EGFand EGFR; PDGF and PDGFR; NGF and NGFR; and IGF and IGFR.

FIG. 3A provides the Loop 1, Loop 2, and Loop 5 amino acid sequences ofwild type EETI, VEGF_CKP9 (also referred to as E9), VEGF_CKP9.54 (alsoreferred to as EM54), and VEGF_CKP9.54.90 (also referred to asV_L2.9.54.90). FIG. 3B shows the results of experiments performed todetermine whether VEGF_CKP9.54.90, VEGF_CKP9.54, and VEGF_CKP9.63.12inhibit trypsin protease activity.

FIG. 4A provides schematics that compare the disulfide bond connectivitypattern of VEGF_CKP9.54.90 (also referred to as 54.90) to that of wildtype EETI-II. FIG. 4B provides the Loop 1, Loop2, and Loop 5 amino acidsequences of VEGF_CKP9 (also referred to as E9), VEGF_CKP9.54 (alsoreferred to as EM54), VEGF_CKP9.54.1 (also referred to as V_L2.9.54.1),VEGF_CKP9.54.90 (also referred to as V_L2.9.54.90), VEGF_CKP9.63 (alsoreferred to as EM63), VEGF_CKP9.63.1 (also referred to as V_L2.9.63.1),VEGF_CKP9.63.44 (also referred to as V_L2.9.63.44), and VEGF_CKP9.63.12(also referred to as V_L2.9.63.12). FIG. 4C shows the results ofexperiments performed to determine whether VEGF_CKP9.54.90 andVEGF_CKP9.63.12 are resistant to trypsin digestion.

FIG. 5A depicts the structure of VEGF_CKP9.54 (also referred to as EM54)superimposed upon the structure of VEGF_CKP9.54 (also referred to asEM63) and provides schematics of VEGF_CKP9.54's and VEGF_CKP9.63'sdisulfide bond connectivity patterns. FIG. 5B depicts the structure ofwild type EETI-II, and provides a schematic of wild type EETI'sdisulfide bond connectivity pattern.

FIG. 6A depicts the co-crystal structure of VEGF_CKP9.54.90 in complexwith VEGF-A. FIG. 6B depicts the co-crystal structure of VEGF_CKP9.54.90in complex with VEGF-A rotated 90° relative to FIG. 6A.

FIG. 7A is a ribbon diagram model showing the binding interface ofVEGF_CKP9.54.90 on VEGF-A. FIG. 7B depicts space a filling model of FIG.7A. FIG. 7C is a ribbon diagram model showing the binding interface ofantibody G6.31 on VEGF-A. FIG. 7D depicts space a filling model of FIG.7C. FIG. 7E is a ribbon diagram model showing the binding interface ofdomain 2 of Flt-2 on VEGF-A. FIG. 7F depicts space a filling model ofFIG. 7E.

FIG. 8A depicts a ribbon diagram model that shows the binding interfaceof VEGF_CKP9.54.90, on VEGF-A. FIG. 8B depicts a ribbon diagram modelthat shows the binding interface of antibody G6.31 on VEGF-A. FIG. 8Cdepicts a ribbon diagram model that shows the binding interface ofdomain 2 of Flt-2 on VEGF-A.

FIG. 9 shows contact residues on VEGF-A at the interacting surfacebetween VEGF-A and VEGF_CKP9.54.90.

FIG. 10A shows the binding interface of bevacizumab Fab on VEGF-A. FIG.10B shows the binding interface of Z-domain on VEGF-A. FIG. 10C showsthe binding interface of receptor-blocking peptide v108 on VEGF-A.

FIG. 11A provides the results of an experiment that was performed todetermine the effects of amino acid substitution mutations in VEGF-A onbinding of VEGF_CKP9.54.90 to VEGF-A and on the binding ofVEGF_CKP9.63.12 to VEGFA. FIG. 11B provides the results of FIG. 11A on adifferent y axis.

FIG. 12 provides the results of experiments that were performed todetermine the effects of VEGF_CKP9.54.90 on CNV in rat eyes.

FIG. 13 provides the results of experiments that were performed todetermine the IC₅₀ values of VEGF-binding CKP variants.

FIG. 14A provides the amino acid sequences of VEGF_CKP9 (also referredto as E9), VEGF_CKP9.54 (also referred to as EM54), and VEGF_CKP9.63(also referred to as EM63). FIG. 14B depicts the structure ofVEGF_CKP9.54. FIG. 14C depicts the structure of VEGF_CKP9.63.

FIG. 14D provides a portion of the co-crystal structure of VEGF_CKP9.63in complex with VEGF-A superimposed on the co-crystal structure ofVEGF_CKP9.54 in complex with VEGF-A. FIG. 14D shows that residue atposition 8 within loopl of VEGF_CKP9.63 could form a hydrogen bond withthe side chain of Gln22 of VEGF-A.

FIG. 15 shows the results of phage competition ELISA experiments thatwere performed to assess the binding affinity of clones 9.54-28, 9.54,9.54.1-2, 9.54.1-36, 9.54.1-42, 9.54.1-63, 9.54.1-90, and 9.54.1 forhVEGF(8-109).

FIG. 16A shows the results of phage competition ELISA experiments thatwere performed to assess the binding affinity of clones 9.63.44-1 to9.63.44-7.

FIG. 16B shows the results of phage competition ELISA experiments thatwere performed to assess the binding affinity of clones 9.63.44-8 to9.63.44-14.

DETAILED DESCRIPTION OF THE INVENTION

Provided are non-naturally occurring cystine knot peptides (CKPs) thatspecifically bind human VEGF-A. Such non-naturally occurring CKPsdemonstrate one or more of the following characteristics: inhibition ofVEGF-A activity with and IC₅₀ between less than about 0.5 nM and lessthan about 1.0 nM; binding to human VEGF-A, mouse VEGF-A, and ratVEGF-A; resistance to trypsin digestion; a disulfide bond connectivityof C1-C4, C2-C3, and C5-C6; an alpha helix content of at least about atleast about 15% to least about 50%; binding to an epitope on VEGF-A thatis different from the epitope bound by antibody G6.31, binding to anepitope on VEGF-A that is different from the epitope bound bybevacizumab, and/or binding to an epitope on VEGF-A that is differentfrom the epitope bound by Flt-1.

Also provided are chimeric molecules and conjugates comprisingnon-naturally occurring cystine knot peptides that bind VEGF-A, nucleicacids encoding non-naturally occurring CKPs that bind VEGF-A, andcompositions (such as pharmaceutical compositions). Also provided aremethods of using non-naturally occurring CKPs that bind VEGF-A fortreating ocular diseases and/or disorders (such as ocular vascularproliferative diseases and/or disorders) resulting from abnormal (suchas excessive) angiogenesis and/or abnormal vascular permeability. Alsoprovided are uses of non-naturally occurring CKPs that bind VEGF-A inthe manufacture of a medicament for the treatment of ocular disease ordisorders.

In a related aspect, non-naturally occurring CKPs that bind human lowdensity lipoprotein receptor-related protein 6 (LRP6) are also provided.

Practice of the present disclosure employs, unless otherwise indicated,standard methods and conventional techniques in the fields of cellbiology, toxicology, molecular biology, biochemistry, cell culture,immunology, oncology, recombinant DNA and related fields as are withinthe skill of the art. Such techniques are described in the literatureand thereby available to those of skill in the art. See, for example,Alberts, B. et al., “Molecular Biology of the Cell,” 5^(th) edition,Garland Science, New York, N.Y., 2008; Voet, D. et al. “Fundamentals ofBiochemistry: Life at the Molecular Level,” 3^(rd) edition, John Wiley &Sons, Hoboken, N.J., 2008; Sambrook, J. et al., “Molecular Cloning: ALaboratory Manual,” 3^(rd) edition, Cold Spring Harbor Laboratory Press,2001; Ausubel, F. et al., “Current Protocols in Molecular Biology,” JohnWiley & Sons, New York, 1987 and periodic updates; Freshney, R. I.,“Culture of Animal Cells: A Manual of Basic Technique,” 4^(th) edition,John Wiley & Sons, Somerset, N J, 2000; and the series “Methods inEnzymology,” Academic Press, San Diego, Calif.

DEFINITIONS

As used herein “non-naturally occurring” means, e.g., a polypeptidecomprising an amino acid sequence that is not found in nature, or, e.g.,a nucleic acid comprising a nucleotide sequence that is not found innature. A “non-naturally occurring cystine knot peptide” or“non-naturally occurring CKP” (or a nucleic acid encoding the same)provided herein does not have the amino acid sequence of a wild typeEETI-II protein, i.e., GCPRILMRCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 1),wherein Loop 1 (L1) is the amino acid sequence PRILMR (SEQ ID NO: 92),Loop 2 (L2) is the amino acid sequence KQDSD (SEQ ID NO: 93), Loop 3(L3) is the amino acid sequence LAG, Loop 4 (L4) is the amino acid V,and Loop 5 (L5) is the amino acid sequence GPNGF (SEQ ID NO: 15). Anon-naturally occurring CKP provided herein can be produced by geneticengineering methods or by chemical synthesis methods. Thus, anon-naturally occurring CKP described herein may be recombinant, i.e.,produced by a cell, or nucleic acid, or vector, that has been modifiedby the introduction of a heterologous nucleic acid or the alteration ofa native nucleic acid to a form not native to that cell, or that thecell is derived from a cell so modified. Alternatively, a non-naturallyoccurring CKP described herein can be produced via chemical peptidesynthesis.

As used herein, the term “cystine-knot peptide” or “CKP” refers to apeptide between 26-50 amino acids in length, which contain six conservedcysteine residues that form three disulfide bonds. One of the disulfidespenetrates the macrocycle which is formed by the two other disulfidesand their interconnecting backbones, thereby yielding a characteristicknotted topology with multiple loops exposed on the surface. The loopsare defined as the amino acid regions which flank the six conservedcysteine residues and are highly variable in nature.

As used herein, an “amino acid alteration” refers to the addition,deletion, or substitution of at least one amino acid in, e.g., a peptidesequence (such as in the WT EETI-II peptide sequence to generate anon-naturally occurring CKP, or in a non-naturally occurring CKP togenerate another non-naturally occurring CKP).

An “isolated” non-naturally occurring CKP or composition is one whichhas been identified and separated and/or recovered from a component ofits natural environment. Contaminant components of its naturalenvironment are materials which would interfere with diagnostic ortherapeutic uses for the non-naturally occurring CKP, and can includeenzymes, hormones, and other proteinaceous or nonproteinaceous solutes.In preferred embodiments, the non-naturally occurring CKP or compositionwill be purified (1) to greater than 95% by weight of non-naturallyoccurring CKP as determined by the Lowry method, and most preferablymore than 99% by weight, (2) to a degree sufficient to obtain at least15 residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (3) to homogeneity by SDS-PAGE underreducing or nonreducing conditions using Coomassie blue or, preferably,silver stain. Isolated non-naturally occurring CKP includes the CKP insitu within recombinant cells since at least one component of the CKP'snatural environment will not be present. An isolated non-naturallyoccurring CKP will be prepared by at least one purification step.

“Percent (%) amino acid sequence identity” or “homology” with respect tothe polypeptide sequences identified herein is defined as the percentageof amino acid residues in a candidate sequence that are identical withthe amino acid residues in the polypeptide being compared, afteraligning the sequences considering any conservative substitutions aspart of the sequence identity. Alignment for purposes of determiningpercent amino acid sequence identity can be achieved in various waysthat are within the skill in the art, for instance, using publiclyavailable computer software such as BLAST, BLAST-2, ALIGN or Megalign(DNASTAR) software. Those skilled in the art can determine appropriateparameters for measuring alignment, including any algorithms needed toachieve maximal alignment over the full length of the sequences beingcompared. For purposes herein, however, % amino acid sequence identityvalues are generated using the sequence comparison computer programALIGN-2. The ALIGN-2 sequence comparison computer program was authoredby Genentech, Inc. and the source code has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. The ALIGN-2 program should be compiled for use ona UNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

As used herein the term “epitope” refers to a protein determinantcapable of being specifically bound by a non-naturally occurring CKPprovided herein. An epitope can comprise between about 3-10 amino acidsin a spatial conformation, which is unique to the epitope. These aminoacids can be linear within the protein (i.e., consecutive in the aminoacid sequence) or they can be positioned in different parts of theprotein (i.e., non-consecutive in the amino acid sequence). Methods ofdetermining the spatial conformation of amino acids within a protein, orat the interface of two proteins, are known in the art, and include, forexample, x-ray crystallography and 2-dimensional nuclear magneticresonance.

The terms “disulfide bonding pattern (DBP),” “disulfide bondconnectivity,” and “disulfide linkage pattern” refers to the linkingpattern of the cysteines relative to the WT EETI-II protein. The WTEETI-II protein comprises six conserved cysteine residues (numbered 1-6)that form three disulfide bonds with connectivities C1-C4, C2-05, andC3-C6. The disulfide bonding pattern is topologically constant, meaningthe disulfide bonds can only be changed by unlinking one or moredisulfides such as using redox conditions.

A “subject,” “patient,” or an “individual” for purposes of treatmentrefers to any animal classified as a mammal, including humans, domesticand farm animals, and zoo, sports, or pet animals, such as dogs, horses,cats, cows, etc. Preferably, the mammal is human.

An “effective amount” of a non-naturally occurring CKP (or a compositioncomprising such a non-naturally occurring CKP) as disclosed herein is anamount sufficient to carry out a specifically stated purpose. An“effective amount” can be determined empirically and by known methodsrelating to the stated purpose.

The term “therapeutically effective amount” refers to an amount of anon-naturally occurring CKP or composition as disclosed herein,effective to “treat” a disease or disorder in a mammal (such as a humanpatient). In the case of ocular disease or ocular disorder (such as anocular vascular proliferative disease or ocular disorder characterizedby excessive angiogenesis), the therapeutically effective amount of anon-naturally occurring CKP that binds VEGF-A described herein (or acomposition comprising such a non-naturally occurring VEGF-A-bindingCKP) refers to the amount to reduce, stop or prevent at least onesymptom of the ocular disease, such as a symptom or disorder of anocular disease described in further detail elsewhere herein. Forexample, an effective amount would be considered as the amountsufficient to reduce or prevent a symptom of the ocular disease orocular disorder (such as an ocular vascular proliferative disease orocular disorder characterized by excessive angiogenesis), for example acomplete or partial resolution and/or maintenance of the ocular diseaseas measured by optical coherence tomography (OCT) or an increase and/ormaintenance in best corrected visual acuity (such as greater than 5letters as assessed by EDTRS eye chart), or a reduction in the size ofthe neovascularization or neovascular permeability as assessed by fundusfluorescence angiography. An effective amount used herein would alsoinclude an amount sufficient to prevent or delay the development of,e.g., macular edema, enhanced permeability (such as retinal vascularpermeability), size of CNV lesion, and vision loss. An effective amountas used herein would also include an amount sufficient to prevent ordelay the development of a symptom of the ocular disease, alter thecourse of a symptom disease (for example but not limited to, slow theprogression of a symptom of the ocular disease), or reverse a symptom ofthe disease.

As used herein, “treatment” or “treating” is an approach for obtainingbeneficial or desired results including clinical results. For purposesof this invention, beneficial or desired clinical results include, butare not limited to, one or more of the following: alleviating one ormore symptoms resulting from the ocular disease, diminishing the extentof the ocular disease, stabilizing the ocular disease (e.g., preventingor delaying the worsening of the disease), preventing or delaying thespread of the disease (such as to surrounding ocular tissues),preventing or delaying the recurrence of the ocular disease, delay orslowing the progression of the ocular disease, ameliorating the diseasestate, providing a remission or resolution (partial or total) of theocular disease, decreasing the dose of one or more other medicationsrequired to treat the ocular disease, delaying the progression of theocular disease, increasing or improving the quality of life, and/orpreventing or delaying vision loss. Also encompassed by “treatment” is areduction of pathological consequence of an ocular disease (such as, forexample, vision loss). The methods provided herein contemplate any oneor more of these aspects of treatment.

A “disorder” is any condition that would benefit from treatment with anon-naturally occurring CKP that binds VEGF-A described herein.Non-limiting examples of VEGF-A-related disorders to be treated hereininclude ocular diseases and disorders (such as ocular vascularproliferative diseases or ocular disorders characterized by excessiveangiogenesis), as described elsewhere herein.

As used herein, by “pharmaceutically acceptable” or “pharmacologicallycompatible” is meant a material that is not biologically or otherwiseundesirable, e.g., the material may be incorporated into apharmaceutical composition administered to a patient without causing anysignificant undesirable biological effects or interacting in adeleterious manner with any of the other components of the compositionin which it is contained. Pharmaceutically acceptable carriers orexcipients have preferably met the required standards of toxicologicaland manufacturing testing and/or are included on the Inactive IngredientGuide prepared by the U.S. Food and Drug administration.

The term “detecting” is intended to include determining the presence orabsence of a substance or quantifying the amount of a substance (such asa target ligand). The term thus refers to the use of the materials,compositions, and methods provided herein for qualitative andquantitative determinations. In general, the particular technique usedfor detection is not critical for practice of the invention.

For example, “detecting” according to the invention may include:observing the presence or absence of a target ligand (including, but notlimited to, a human low density lipoprotein receptor-related protein 6(LRP6) polypeptide or a human vascular endothelial growth factor A(VEGF-A) polypeptide); a change in the levels of a target ligand; and/ora change in biological function/activity of a target ligand. In certainembodiments, “detecting” may include detecting levels of a target ligand(e.g., polypeptide levels of a human LRP6 or a human VEGF-A). Detectingmay include quantifying a change (increase or decrease) of any valuebetween 10% and 90%, or of any value between 30% and 60%, or over 100%,when compared to a control. Detecting may include quantifying a changeof any value between 2-fold to 10-fold, inclusive, or more e.g.,100-fold.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to thenon-naturally occurring CKP. The label may itself be detectable byitself (e.g., radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

With regard to the binding of a non-naturally occurring CKP to a targetligand, the term “specific binding” or “specifically binds to” or is“specific for” a particular target ligand means that binding that ismeasurably different from a non-specific interaction. Specific bindingcan be measured, for example, by determining binding of a moleculecompared to binding of a control molecule, which generally is a moleculeof similar structure that does not have binding activity. For example,specific binding can be determined by competition with a controlmolecule that is similar to the target, for example, an excess ofnon-labeled target. In this case, specific binding is indicated if thebinding of the labeled target to a probe is competitively inhibited byexcess unlabeled target. In certain embodiments, the extent of bindingof the non-naturally occurring CKP to a “non-target” ligand will be lessthan about 10% of the binding of the non-naturally occurring CKP to itstarget ligand (such as LRP6 or VEGF-A) as determined by, e.g.,fluorescence activated cell sorting (FACS) analysis orradioimmunoprecipitation (RIA). In certain embodiments, a non-naturallyoccurring CKP of the present disclosure specifically binds to a targetligand (such as human low density lipoprotein receptor-related protein 6(LRP6) or human vascular endothelial growth factor A (VEGF-A)) with adissociation constant (Kd) equal to or lower than 100 nM, optionallylower than 10 nM, optionally lower than 1 nM, optionally lower than 0.5nM, optionally lower than 0.1 nM, optionally lower than 0.01 nM, oroptionally lower than 0.005 nM; measured at a temperature of about 4°C., 25° C., 37° C., or 45° C.

Reference to “about” a value or parameter herein refers to the usualerror range for the respective value readily known to the skilled personin this technical field. Reference to “about” a value or parameterherein includes (and describes) aspects that are directed to that valueor parameter per se. For example, description referring to “about X”includes description of “X.”

It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

All references cited herein, including patent applications andpublications, are hereby incorporated by reference in their entirety.

Non-Naturally Occurring Cystine Knot Peptides (CKPs) that Bind HumanVascular Endothelial Growth Factor A (VEGF-A)

In certain embodiments, provided herein is a non-naturally occurringcystine knot peptide (CKP) that binds to vascular endothelial growthfactor A (VEGF-A), wherein the CKP comprises the following cystinescaffold structure (i.e., scaffold structure I):

Z₁C₁L1C₂L2C₃L3C₄L4C₅L5C₆Z₂  (I)

wherein:

-   -   Z₁ and Z₂ are any amino acid;    -   L1 is Loop 1 and has a structure selected from the group        consisting of: X₁X₂X₃X₄X₅X₆X₇X₈, X₁X₂X₃X₄X₅X₆X₇X₈X₉, and        X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀, wherein each of X₁-X₁₀ is any amino acid;

L2 is Loop 2 and has the structure: X₁X₂X₃X₄X₅, wherein each of X₁-X₅ isany amino acid or an unnatural amino acid selected from the groupconsisting of L-propargylglycine-PEG₆-, L-sulfotyrosine, L-norleucine,L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan,L-3-fluorotyrosine, L-4-fluorophenylalanine, gamma-benzyl-L-proline,gamma-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline,4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine,3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine,3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NHmeta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine,3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine,2-naphthyl-L-alanine, L-4,4′-biphenylalanine,3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine,3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine,4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine,benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine(also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine,cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine(also known as L-tert-Leucine), L-cyclopentylglycine,L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine,3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine,2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine,4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine,L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester ofglycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, andglycine morpholine amide.

L3 is Loop 3 and has the structure: X₁X₂X₃ wherein each of X₁-X₃ is anyamino acid or an unnatural amino acid selected from the group consistingof L-propargylglycine-PEG₆-, L-sulfotyrosine, L-norleucine,L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan,L-3-fluorotyrosine, L-4-fluorophenylalanine, gamma-benzyl-L-proline,gamma-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline,4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine,3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine,3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NHmeta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine,3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine,2-naphthyl-L-alanine, L-4,4′-biphenylalanine,3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine,3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine,4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine,benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine(also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine,cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine(also known as L-tert-Leucine), L-cyclopentylglycine,L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine,3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine,2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine,4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine,L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester ofglycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, andglycine morpholine amide;

L4 is Loop 4 and has the structure: X₁, wherein X₁ is any amino acid oran unnatural amino acid selected from the group consisting ofL-propargylglycine-PEG₆-, L-sulfotyrosine, L-norleucine,L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan,L-3-fluorotyrosine, L-4-fluorophenylalanine, gamma-benzyl-L-proline,gamma-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline,4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine,3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine,3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NHmeta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine,3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine,2-naphthyl-L-alanine, L-4,4′-biphenylalanine,3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine,3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine,4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine,benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine(also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine,cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine(also known as L-tert-Leucine), L-cyclopentylglycine,L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine,3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine,2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine,4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine,L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester ofglycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, andglycine morpholine amide;

L5 is Loop 5 and has the structure: X₁X₂X₃X₄X₅, wherein each of X₁-X₅ isany amino acid or an unnatural amino acid selected from the groupconsisting of L-propargylglycine-PEG₆-, L-sulfotyrosine, L-norleucine,L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan,L-3-fluorotyrosine, L-4-fluorophenylalanine, gamma-benzyl-L-proline,gamma-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline,4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine,3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine,3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NHmeta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine,3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine,2-naphthyl-L-alanine, L-4,4′-biphenylalanine,3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine,3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine,4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine,benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine(also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine,cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine(also known as L-tert-Leucine), L-cyclopentylglycine,L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine,3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine,2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine,4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine,L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester ofglycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, andglycine morpholine amide; and

wherein the CKP binds to VEGF-A with an affinity of 500 pM or less.

In certain embodiments, the C-terminus of the non-naturally occurringcystine knot peptide (CKP) that binds to VEGF-A is modified (such ascapped). In certain embodiments, the N-terminus of the non-naturallyoccurring cystine knot peptide (CKP) that binds to VEGF-A is modified(such as capped). In certain embodiments, both the C- and N-termini ofthe non-naturally occurring cystine knot peptide (CKP) that binds toVEGF-A are modified (such as capped). In certain embodiments, theC-terminal carboxyl group of the non-naturally occurring cystine knotpeptide (CKP) that binds to VEGF-A is amidated. In certain embodiments,the N-terminal amine of the non-naturally occurring cystine knot peptide(CKP) that binds to VEGF-A is acetylated. In certain embodiments, theC-terminal carboxyl group of the non-naturally occurring cystine knotpeptide (CKP) that binds to VEGF-A is amidated and the N-terminal amineof the non-naturally occurring cystine knot peptide (CKP) that binds toVEGF-A is acetylated.

In certain embodiments, the non-naturally occurring cystine knot peptide(CKP) that binds to VEGF-A has an altered disulfide bondconnectivity—with reference to a wild-type Ecballium elaterium trypsininhibitor EETI-II protein having the amino acid sequence set forth inSEQ ID NO: 1; wherein the altered disulfide bond connectivity is C1-C4,C2-C3 and C5-C6.

In certain embodiments, Z₁ and/or Z₂ of the non-naturally occurringcystine knot peptide (CKP) that binds to VEGF-A is G. In certainembodiments, Z₁ and/or Z₂ comprise more than one amino acid. In certainembodiments, Z₁ and/or Z₂ comprise 4 amino acids. In certainembodiments, Z₁ and/or Z₂ comprise 5 amino acids. In certainembodiments, Z₁ and/or Z₂ is an unnatural amino acid. In certainembodiments, the unnatural amino acid is N-acetylglycine or glycineamide. In certain embodiments, the non-naturally occurring cystine knotpeptide (CKP) that binds to VEGF-A comprises an L1 wherein X₃ is not I;wherein X₅ is not M; and/or wherein X₆ is not R. In certain embodiments,the non-naturally occurring cystine knot peptide (CKP) that binds toVEGF-A comprises an L1 wherein X₁ is an amino acid selected from P, Q,R, T, V, D, N, K, L, and X; wherein X₂ is an amino acid selected from T,D, L, V, I, R, P, N and X; wherein X₃ is an amino acid selected from T,P, M, L, S, F, R, and X; wherein X₄ is an amino acid selected from R, T,Q, D, W, L, E, S, K, and X; wherein X₅ is an amino acid selected from F,P, V, E, K, L, I, and X; wherein X₆ is an amino acid selected from K, N,F, P, L, Y, T, D, M, and X; wherein X₇ is an amino acid selected from Q,W, H and/X; and/or wherein X₈ is an amino acid selected from Y, A, G, D,E, W, S, and X, wherein X is and unnatural amino acid is selected fromthe group consisting of L-propargylglycine-PEG₆-, L-sulfotyrosine,L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine,L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine,gamma-benzyl-L-proline, gamma-(4-fluoro-benzyl)-L-proline,4-OH-L-proline, 4-fluoro-L-proline,4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine,3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine,3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NHmeta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine,3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine,2-naphthyl-L-alanine, L-4,4′-biphenylalanine,3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine,3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine,4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine,benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine(also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine,cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine(also known as L-tert-Leucine), L-cyclopentylglycine,L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine,3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine,2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine,4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine,L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester ofglycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, andglycine morpholine amide. In certain embodiments, the non-naturallyoccurring cystine knot peptide (CKP) that binds to VEGF-A comprises anL1 wherein X₉ is an amino acid selected from L, I, V, D, E and X,wherein X is and unnatural amino acid is selected from the groupconsisting of L-propargylglycine-PEG₆-, L-sulfotyrosine, L-norleucine,L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan,L-3-fluorotyrosine, L-4-fluorophenylalanine, gamma-benzyl-L-proline,gamma-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline,4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine,3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine,3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NHmeta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine,3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine,2-naphthyl-L-alanine, L-4,4′-biphenylalanine,3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine,3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine,4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine,benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine(also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine,cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine(also known as L-tert-Leucine), L-cyclopentylglycine,L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine,3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine,2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine,4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine,L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester ofglycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, andglycine morpholine amide. In certain embodiments, the non-naturallyoccurring cystine knot peptide (CKP) that binds to VEGF-A comprises anL1 wherein X₁₀ is an amino acid selected from Y, T, M, N, F, and X,wherein X is and unnatural amino acid is selected from the groupconsisting of L-propargylglycine-PEG₆-, L-sulfotyrosine, L-norleucine,L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan,L-3-fluorotyrosine, L-4-fluorophenylalanine, gamma-benzyl-L-proline,gamma-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline,4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine,3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine,3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NHmeta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine,3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine,2-naphthyl-L-alanine, L-4,4′-biphenylalanine,3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine,3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine,4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine,benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine(also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine,cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine(also known as L-tert-Leucine), L-cyclopentylglycine,L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine,3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine,2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine,4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine,L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester ofglycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, andglycine morpholine amide.

In certain embodiments, the non-naturally occurring cystine knot peptide(CKP) that binds to VEGF-A comprises an L5 wherein each of X₁-X₅ is anyamino acid or an unnatural amino acid selected from the group consistingof L-propargylglycine-PEG₆-, L-sulfotyrosine, L-norleucine,L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan,L-3-fluorotyrosine, L-4-fluorophenylalanine, gamma-benzyl-L-proline,gamma-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline,4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine,3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine,3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NHmeta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine,3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine,2-naphthyl-L-alanine, L-4,4′-biphenylalanine,3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine,3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine,4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine,benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine(also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine,cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine(also known as L-tert-Leucine), L-cyclopentylglycine,L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine,3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine,2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine,4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine,L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester ofglycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, andglycine morpholine amide; with the exception that X₂ is not proline (P).

In certain embodiments, the non-naturally occurring cystine knot peptide(CKP) that binds to VEGF-A comprises an L5 wherein each of X₁-X₅ is anyamino acid or an unnatural amino acid selected from the group consistingof L-propargylglycine-PEG₆-, L-sulfotyrosine, L-norleucine,L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan,L-3-fluorotyrosine, L-4-fluorophenylalanine, gamma-benzyl-L-proline,gamma-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline,4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine,3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine,3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NHmeta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine,3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine,2-naphthyl-L-alanine, L-4,4′-biphenylalanine,3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine,3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine,4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine,benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine(also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine,cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine(also known as L-tert-Leucine), L-cyclopentylglycine,L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine,3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine,2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine,4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine,L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester ofglycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, andglycine morpholine amide, with the exception that X₄ is not glycine (G).In certain embodiments, the non-naturally occurring cystine knot peptide(CKP) that binds to VEGF-A comprises an L5 wherein X₁ is an amino acidselected from G, Q, H, R, L, and Q; wherein X₂ is an amino acid selectedfrom P, M, W, Y, F, L, and H; wherein X₃ is an amino acid selected fromN, F, H, and Y; wherein X₄ is an amino acid selected from G, Q, D, N, K,H, E, and S; and/or wherein X₅ is an amino acid selected from F, S, andT. In certain embodiments, the non-naturally occurring cystine knotpeptide (CKP) that binds to VEGF-A comprises an L2 wherein X₁ is K, X₂is Q, X₃ is D, X₄ is S, and X₅ is D. In certain embodiments, thenon-naturally occurring cystine knot peptide (CKP) that binds to VEGF-Acomprises an L3 wherein X₁ is L, X₂ is A, and X₃ is G. In certainembodiments, the non-naturally occurring cystine knot peptide (CKP) thatbinds to VEGF-A comprises an L4 wherein X₁ is V or F.

In certain embodiments, the non-naturally occurring cystine knot peptide(CKP) that binds to VEGF-A comprises an L1 comprising the structureX₁X₂X₃X₄X₅X₆X₇X₈, wherein: X₁ is an amino acid selected from P, Q, andR; X₂ is an amino acid selected from T, L, and D; X₃ is an amino acidselected from T, M and L; X₄ is an amino acid selected from R, Q, and D;X₅ is an amino acid selected from F, P, and V; X₆ is an amino acidselected from K and F; X₇ is an amino acid selected from Q and W; and X₈is an amino acid selected from Y, G, and D. In certain embodiments, thenon-naturally occurring cystine knot peptide (CKP) that binds to VEGF-Acomprises an L1 comprising the structure X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀, whereinX₁ is an amino acid selected from Q, R, T and V; X₂ is an amino acidselected from T and D; X₃ is P; X₄ is an amino acid selected from T andW; X₅ is an amino acid selected from F, E, P, and K; X₆ is an amino acidselected from N and P; X₇ is an amino acid selected from W and H; X₈ isan amino acid selected from A, D, E, and W; X₉ is an amino acid selectedfrom L and I; and X₁₀ is an amino acid selected from Y, T, M and N. Incertain embodiments, the non-naturally occurring cystine knot peptide(CKP) that binds to VEGF-A comprises an L5 wherein X₁ is an amino acidselected from G, H, and Q; X₂ is an amino acid selected from P, M, W,and Y; X₃ is an amino acid selected from N and Y; X₄ is an amino acidselected from G, Q, and S; and X₅ is an amino acid selected from F andS. In certain embodiments, the non-naturally occurring cystine knotpeptide (CKP) that binds to VEGF-A comprises an L2 wherein X₁ is K, X₂is Q, X₃ is D, X₄ is S, and X₅ is D. In certain embodiments, thenon-naturally occurring cystine knot peptide (CKP) that binds to VEGF-Acomprises an L3 wherein X₁ is L, X₂ is A, and X₃ is G. In certainembodiments, the non-naturally occurring cystine knot peptide (CKP) thatbinds to VEGF-A comprises an L4 wherein X₁ is V or F.

In certain embodiments, the non-naturally occurring cystine knot peptide(CKP) that binds to VEGF-A comprises an L1 having the structureX₁X₂X₃X₄X₅X₆X₇X₈, wherein: X₁ is an amino acid selected from D, Q, N,and K; X₂ is an amino acid selected from V, I, R, L, and P; X₃ is anamino acid selected from L, S, M, T, and F; X₄ is an amino acid selectedfrom Q, L, and E; X₅ is P; X₆ is an amino acid selected from F, L, andY; X₇ is W; and X₈ is G. In certain embodiments, the non-naturallyoccurring cystine knot peptide (CKP) that binds to VEGF-A comprises anL5 wherein X₃ is Y; X₅ is 5; and wherein X₁, X₂ and X₄ are each anyamino acid, with the exception that X₁ is not G, X₂ is not P, X₄ is notG, and/or X₅ is not F. In certain embodiments, the non-naturallyoccurring cystine knot peptide (CKP) that binds to VEGF-A comprises anL5 wherein X₁ is an amino acid selected from H, L, R, and Q; X₂ is anamino acid selected from W, F, and Y; X₃ is Y; X₄ is an amino acidselected from Q, N, K, H, and E; and X₅ is S. In certain embodiments,the non-naturally occurring cystine knot peptide (CKP) that binds toVEGF-A comprises an L2 wherein X₁ is K, X₂ is Q, X₃ is D, X₄ is S, andX₅ is D. In certain embodiments, the non-naturally occurring cystineknot peptide (CKP) that binds to VEGF-A comprises an L3 wherein X₁ is L,X₂ is A, and X₃ is G. In certain embodiments, the non-naturallyoccurring cystine knot peptide (CKP) that binds to VEGF-A comprises anL4 wherein X₁ is V or F.

In certain embodiments, the non-naturally occurring cystine knot peptide(CKP) that binds to VEGF-A comprises an L1 comprising the structureX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀, wherein X₁ is an amino acid selected from K, Q,L, and R; X₂ is an amino acid selected from N and D; X₃ is an amino acidselected from P and L; X₄ is an amino acid selected from L, T, S and K;X₅ is an amino acid selected from F, V, I, and L; X₆ is an amino acidselected from N and D; X₇ is W; X₈ is an amino acid selected from A andS; X₉ is an amino acid selected from L, V, E and D; and X₁₀ is an aminoacid selected from Y and F. In certain embodiments, the non-naturallyoccurring cystine knot peptide (CKP) that binds to VEGF-A comprises anL5 wherein X₁ is Q; X₂ is an amino acid selected from L, F, M, and H; X₃is an amino acid selected from F, Y, and H; X₄ is an amino acid selectedfrom D, Q, N, and K; and X₅ is an amino acid selected from S and T. Incertain embodiments, the non-naturally occurring cystine knot peptide(CKP) that binds to VEGF-A comprises an L2 wherein X₁ is K, X₂ is Q, X₃is D, X₄ is S, and X₅ is D. In certain embodiments, the non-naturallyoccurring cystine knot peptide (CKP) that binds to VEGF-A comprises anL3 wherein X₁ is L, X₂ is A, and X₃ is G. In certain embodiments, thenon-naturally occurring cystine knot peptide (CKP) that binds to VEGF-Acomprises an L4 wherein X₁ is V or F.

In certain embodiments, the non-naturally occurring cystine knot peptide(CKP) that binds to VEGF-A comprises an L1 comprising the structureX₁X₂X₃X₄X₅X₆X₇X₈, wherein: X₅ is P; X₇ is W; X₈ is G; and wherein X₁,X₂, X₃, X₄ and X₆ are each any amino acid, with the exception that X₁ isnot P, X₂ is not R, X₃ is not I, and/or X₆ is not R. In certainembodiments, the non-naturally occurring cystine knot peptide (CKP) thatbinds to VEGF-A comprises an L1 comprising the structureX₁X₂X₃X₄X₅X₆X₇X₈, wherein X₁ is an amino acid selected from N and D; X₂is an amino acid selected from I and V; X₃ is an amino acid selectedfrom M and L; X₄ is an amino acid selected from L, Q, D and K; X₅ is P;X₆ is an amino acid selected from F, Y, T, L, and M; X₇ is W; and X₈ isG. In certain embodiments, the non-naturally occurring cystine knotpeptide (CKP) that binds to VEGF-A comprises an L5 wherein X₁ is anamino acid selected from Q, H, L, and R; X₂ is an amino acid selectedfrom Y and W; X₃ is Y; X₄ is an amino acid selected from Q and N; and X₅is S. In certain embodiments, the non-naturally occurring cystine knotpeptide (CKP) that binds to VEGF-A comprises an L5 wherein X₃ is Y; X₅is 5; and wherein X₁, X₂, and X₄ are each any amino acid, with theexception that X₁ is not G, X₂ is not P, and/or X₄ is not G. In certainembodiments, the non-naturally occurring cystine knot peptide (CKP) thatbinds to VEGF-A comprises an L2 wherein X₁ is an amino acid selectedfrom G or E; X₂ is an amino acid selected from Q, L, P, R, E, and M; X₃is an amino acid selected from S, D, and N; X₄ is an amino acid selectedfrom F, Y, L, M, and I; and/or X₅ is an amino acid selected from E, D,Q, L, and S. In certain embodiments, the non-naturally occurring cystineknot peptide (CKP) that binds to VEGF-A comprises an L3 wherein X₁ is L,X₂ is A, and X₃ is G. In certain embodiments, the non-naturallyoccurring cystine knot peptide (CKP) that binds to VEGF-A comprises anL4 wherein X₁ is V or F.

In certain embodiments, the non-naturally occurring cystine knot peptide(CKP) that binds to VEGF-A comprises an L5, wherein each of X₁-X₅ is anyamino acid with the exception that X₂ is not proline (P). In certainembodiments, the non-naturally occurring cystine knot peptide (CKP) thatbinds to VEGF-A comprises an L5, wherein each of X₁-X₅ is any amino acidwith the exception that X₄ is not glycine (G). In certain embodiments,the non-naturally occurring cystine knot peptide (CKP) that binds toVEGF-A comprises an L5, wherein X₁ is any amino acid except G; X₂ is anyamino acid except P; X₃ is any amino acid except N; X₄ is any amino acidexcept G; and/or X₅ is any amino acid except F.

In certain embodiments, the non-naturally occurring cystine knot peptide(CKP) that binds to VEGF-A comprises an L1 comprising the structureX₁X₂X₃X₄X₅X₆X₇X₈, wherein X₁ is an amino acid selected from N, D, and X;X₂ is an amino acid selected from I, V, and X; X₃ is M or X; X₄ is anamino acid selected from L, Q, and X; X₅ is P or X; X₆ is F, Y, or X; X₇is W or X; and X₈ is G or X, wherein X is an unnatural amino acidselected from the group consisting of L-propargylglycine-PEG₆-,L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine,L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine,gamma-benzyl-L-proline, gamma-(4-fluoro-benzyl)-L-proline,4-OH-L-proline, 4-fluoro-L-proline,4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine,3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine,3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NHmeta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine,3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine,2-naphthyl-L-alanine, L-4,4′-biphenylalanine,3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine,3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine,4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine,benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine(also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine,cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine(also known as L-tert-Leucine), L-cyclopentylglycine,L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine,3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine,2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine,4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine,L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester ofglycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, andglycine morpholine amide. In certain embodiments, the non-naturallyoccurring cystine knot peptide (CKP) that binds to VEGF-A comprises anL3 wherein each of X₁-X₃ is any amino acid or an unnatural amino acidselected from the group consisting of L-propargylglycine-PEG₆-,L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine,L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine,gamma-benzyl-L-proline, gamma-(4-fluoro-benzyl)-L-proline,4-OH-L-proline, 4-fluoro-L-proline,4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine,3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine,3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NHmeta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine,3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine,2-naphthyl-L-alanine, L-4,4′-biphenylalanine,3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine,3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine,4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine,benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine(also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine,cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine(also known as L-tert-Leucine), L-cyclopentylglycine,L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine,3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine,2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine,4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine,L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester ofglycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, andglycine morpholine amide, with the exception that X₁ is not Leucine (L),X₂ is not Alanine (A), and X₃ is not glycine (G), wherein the unnaturalamino acid selected from the group consisting ofL-propargylglycine-PEG₆-, L-sulfotyrosine, L-norleucine,L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan,L-3-fluorotyrosine, L-4-fluorophenylalanine, gamma-benzyl-L-proline,gamma-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline,4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine,3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine,3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NHmeta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine,3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine,2-naphthyl-L-alanine, L-4,4′-biphenylalanine,3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine,3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine,4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine,benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine(also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine,cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine(also known as L-tert-Leucine), L-cyclopentylglycine,L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine,3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine,2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine,4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine,L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester ofglycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, andglycine morpholine amide. In certain embodiments, the non-naturallyoccurring cystine knot peptide (CKP) that binds to VEGF-A comprises anL3 wherein X₁ is an amino acid selected from M, F, L V, and X; X₂ is anamino acid selected from S, N, Q, I, Y, E, V, T, and X; and X₃ is anamino acid selected from D, Q, T, N, E, R, and X, wherein X is anunnatural amino acid selected from the group consisting ofL-propargylglycine-PEG₆-, L-sulfotyrosine, L-norleucine,L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan,L-3-fluorotyrosine, L-4-fluorophenylalanine, gamma-benzyl-L-proline,gamma-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline,4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine,3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine,3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NHmeta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine,3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine,2-naphthyl-L-alanine, L-4,4′-biphenylalanine,3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine,3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine,4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine,benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine(also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine,cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine(also known as L-tert-Leucine), L-cyclopentylglycine,L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine,3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine,2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine,4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine,L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester ofglycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, andglycine morpholine amide. In certain embodiments, the non-naturallyoccurring cystine knot peptide (CKP) that binds to VEGF-A comprises anL4 wherein X₁ is any amino acid except V, or an unnatural amino acidselected from the group consisting of L-propargylglycine-PEG₆-,L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine,L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine,gamma-benzyl-L-proline, gamma-(4-fluoro-benzyl)-L-proline,4-OH-L-proline, 4-fluoro-L-proline,4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine,3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine,3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NHmeta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine,3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine,2-naphthyl-L-alanine, L-4,4′-biphenylalanine,3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine,3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine,4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine,benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine(also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine,cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine(also known as L-tert-Leucine), L-cyclopentylglycine,L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine,3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine,2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine,4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine,L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester ofglycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, andglycine morpholine amide. In certain embodiments, the non-naturallyoccurring cystine knot peptide (CKP) that binds to VEGF-A comprises anL4 wherein X₁ is I, L, or X, wherein X is an unnatural amino acidselected from the group consisting of L-propargylglycine-PEG₆-,L-sulfotyrosine, L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine,L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalaninegamma-benzyl-L-proline, gamma-(4-fluoro-benzyl)-L-proline,4-OH-L-proline, 4-fluoro-L-proline,4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine,3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine,3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NHmeta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine,3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine,2-naphthyl-L-alanine, L-4,4′-biphenylalanine,3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine,3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine,4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine,benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine(also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine,cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine(also known as L-tert-Leucine), L-cyclopentylglycine,L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine,3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine,2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine,4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine,L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester ofglycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, andglycine morpholine amide. In certain embodiments, the non-naturallyoccurring cystine knot peptide (CKP) that binds to VEGF-A comprises anL5 wherein X₃ is Y or an unnatural amino acid selected from the groupconsisting of L-propargylglycine-PEG₆-, L-sulfotyrosine, L-norleucine,L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan,L-3-fluorotyrosine, L-4-fluorophenylalanine, gamma-benzyl-L-proline,gamma-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline,4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine,3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine,3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NHmeta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine,3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine,2-naphthyl-L-alanine, L-4,4′-biphenylalanine,3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine,3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine,4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine,benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine(also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine,cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine(also known as L-tert-Leucine), L-cyclopentylglycine,L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine,3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine,2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine,4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine,L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester ofglycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, andglycine morpholine amide; X₅ is S or an unnatural amino acid selectedfrom the group consisting of L-propargylglycine-PEG₆-, L-sulfotyrosine,L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine,L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine,gamma-benzyl-L-proline, gamma-(4-fluoro-benzyl)-L-proline,4-OH-L-proline, 4-fluoro-L-proline,4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine,3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine,3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NHmeta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine,3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine,2-naphthyl-L-alanine, L-4,4′-biphenylalanine,3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine,3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine,4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine,benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine(also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine,cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine(also known as L-tert-Leucine), L-cyclopentylglycine,L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine,3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine,2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine,4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine,L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester ofglycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, andglycine morpholine amide; and wherein X₁, X₂, and X₄ are each any aminoacid or an unnatural amino acid selected from the group consisting ofL-propargylglycine-PEG₆-, L-sulfotyrosine, L-norleucine,L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan,L-3-fluorotyrosine, L-4-fluorophenylalanine, gamma-benzyl-L-proline,gamma-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline,4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine,3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine,3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NHmeta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine,3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine,2-naphthyl-L-alanine, L-4,4′-biphenylalanine,3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine,3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine,4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine,benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine(also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine,cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine(also known as L-tert-Leucine), L-cyclopentylglycine,L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine,3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine,2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine,4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine,L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester ofglycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, andglycine morpholine amide, with the exception that X₁ is not G, X₂ is notP, and/or X₄ is not G. In certain embodiments, the non-naturallyoccurring cystine knot peptide (CKP) that binds to VEGF-A comprises anL5, wherein each of X₁-X₅ is any amino acid with the exception that X₂is not proline (P). In certain embodiments, the non-naturally occurringcystine knot peptide (CKP) that binds to VEGF-A comprises an L5 whereineach of X₁-X₅ is any amino acid with the exception that X₄ is notglycine (G). In certain embodiments, the non-naturally occurring cystineknot peptide (CKP) that binds to VEGF-A comprises an L5 wherein X₁ is anamino acid selected from Q, H, and X; X₂ is an amino acid selected fromY, W, and X; X₃ is Y or X; X₄ is an amino acid selected from Q, N, or X;X₅ is S or X, wherein X is an unnatural amino acid selected from thegroup consisting of L-propargylglycine-PEG₆-, L-sulfotyrosine,L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine,L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine,gamma-benzyl-L-proline, gamma-(4-fluoro-benzyl)-L-proline,4-OH-L-proline, 4-fluoro-L-proline,4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine,3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine,3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NHmeta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine,3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine,2-naphthyl-L-alanine, L-4,4′-biphenylalanine,3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine,3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine,4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine,benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine(also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine,cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine(also known as L-tert-Leucine), L-cyclopentylglycine,L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine,3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine,2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine,4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine,L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester ofglycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, andglycine morpholine amide. In certain embodiments, the non-naturallyoccurring cystine knot peptide (CKP) that binds to VEGF-A comprises anL2 wherein X₁ is G or X; X₂ is R, P, or X; X₃ is D or X; X₄ is F, I, orX; and X₅ is E, D, or X, wherein X is an unnatural amino acid selectedfrom the group consisting of L-propargylglycine-PEG₆-, L-sulfotyrosine,L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine,L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine,gamma-benzyl-L-proline, gamma-(4-fluoro-benzyl)-L-proline,4-OH-L-proline, 4-fluoro-L-proline,4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine,3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine,3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NHmeta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine,3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine,2-naphthyl-L-alanine, L-4,4′-biphenylalanine,3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine,3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine,4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine,benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine(also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine,cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine(also known as L-tert-Leucine), L-cyclopentylglycine,L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine,3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine,2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine,4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine,L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester ofglycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, andglycine morpholine amide.

In certain embodiments, the C-terminus of the non-naturally occurringcystine knot peptide (CKP) that binds to VEGF-A is modified (such ascapped). In certain embodiments, the N-terminus of the non-naturallyoccurring cystine knot peptide (CKP) that binds to VEGF-A is modified(such as capped). In certain embodiments, both the C- and N-termini ofthe non-naturally occurring cystine knot peptide (CKP) that binds toVEGF-A are modified (such as capped). In certain embodiments, theC-terminal carboxyl group of the non-naturally occurring cystine knotpeptide (CKP) that binds to VEGF-A is amidated. In certain embodiments,the N-terminal amine of the non-naturally occurring cystine knot peptide(CKP) that binds to VEGF-A is acetylated. In certain embodiments, theC-terminal carboxyl group of the non-naturally occurring cystine knotpeptide (CKP) that binds to VEGF-A is amidated and the N-terminal amineof the non-naturally occurring cystine knot peptide (CKP) that binds toVEGF-A is acetylated.

Also provided herein is a non-naturally occurring cystine knot peptide(CKP) that binds to VEGF-A, wherein the CKP comprises the cystinescaffold structure provided below (i.e., scaffold structure I):

Z₁C₁L1C₂L2C₃L3C₄L4C₅L5C₆Z₂  (I)

wherein:

Z₁ and Z₂ are any amino acid;

L1 is Loop 1 and has a structure selected from the group consisting of:X₁X₂X₃X₄X₅X₆X₇X₈, X₁X₂X₃X₄X₅X₆X₇X₈X₉, and X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀, whereineach of X₁-X₁₀ is any amino acid;

L2 is Loop 2 and has the structure: X₁X₂X₃X₄X₅, wherein each of X₁-X₅ isany amino acid;

L3 is Loop 3 and has the structure: X₁X₂X₃ wherein each of X₁-X₃ is anyamino acid;

L4 is Loop 4 and has the structure: X₁, wherein X₁ is any amino acid;

L5 is Loop 5 and has the structure: X₁X₂X₃X₄X₅, wherein each of X₁-X₅ isany amino acid; wherein, the CKP has an altered disulfide bondconnectivity with reference to a wild-type Ecballium elaterium trypsininhibitor EETI-II protein having the amino acid sequence set forth inSEQ ID NO: 1; wherein the altered disulfide bond connectivity is C1-C4,C2-C3 and C5-C6; and wherein the CKP has a percent alpha helix contentof at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,or at least about 50%, including any range in between these values.

In certain embodiments, the non-naturally occurring CKP binds to VEGF-Awith an affinity of about 500 pM or less.

In certain embodiments, the binding affinity of the non-naturallyoccurring CKP to VEGF-A is determined via, e.g., surface plasmonresonance or other assays detailed in the Examples below.

In certain embodiments, Z₁ and/or Z₂ of the non-naturally occurringcystine knot peptide (CKP) that binds to VEGF-A is G. In certainembodiments, Z₁ and/or Z₂ comprise more than one amino acid. In certainembodiments, Z₁ and/or Z₂ comprise 4 amino acids. In certainembodiments, Z₁ and/or Z₂ comprise 5 amino acids. In certainembodiments, Z₁ and/or Z₂ is an unnatural amino acid. In certainembodiments, the unnatural amino acid is N-acetylglycine or glycineamide.

In certain embodiments, the C-terminus of the non-naturally occurringcystine knot peptide (CKP) that binds to VEGF-A is modified (such ascapped). In certain embodiments, the N-terminus of the non-naturallyoccurring cystine knot peptide (CKP) that binds to VEGF-A is modified(such as capped). In certain embodiments, both the C- and N-termini ofthe non-naturally occurring cystine knot peptide (CKP) that binds toVEGF-A are modified (such as capped). In certain embodiments, theC-terminal carboxyl group of the non-naturally occurring cystine knotpeptide (CKP) that binds to VEGF-A is amidated. In certain embodiments,the N-terminal amine of the non-naturally occurring cystine knot peptide(CKP) that binds to VEGF-A is acetylated. In certain embodiments, theC-terminal carboxyl group of the non-naturally occurring cystine knotpeptide (CKP) that binds to VEGF-A is amidated and the N-terminal amineof the non-naturally occurring cystine knot peptide (CKP) that binds toVEGF-A is acetylated.

In certain embodiments, the non-naturally occurring CKP that bindsVEGF-A comprises an L5 wherein each of X₁-X₅ is any amino acid with theexception that X₂ is not proline (P). In certain embodiments, thenon-naturally occurring CKP that binds VEGF-A comprises an L5, each ofX₁-X₅ is any amino acid with the exception that X₄ is not glycine (G).In certain embodiments, the non-naturally occurring CKP that bindsVEGF-A inhibits VEGF-A activity with and IC₅₀ of about 0.5 nM to about1.0 nM. In certain embodiments, the degree of inhibition is determinedvia a cellular IC₅₀ assay, as described in further detail in theExamples below.

In certain embodiments, the non-naturally occurring CKP that bindsVEGF-A comprises an L1 comprising the amino acid sequence HMMYDY (SEQ IDNO: 231) or K/P/Q/R-K/T/L/D-W/T/M/L-Q/R/D-W/F/P/V-W/K/F-Y/Q/W-M/Y/G/D(SEQ ID NO: 115) orE/G/P/Q/R/T/V-T/E/A/D-D/T/I/P-W/V/Q/T/W-Y/F/N/E/P/K-P/E/W/N/P-H/Q/K/W/H-Q/F/E/A/D/W-I/L/H-D/W/P/Y/T/M/N(SEQ ID NO: 232), with reference to scaffold structure I above. Incertain embodiments, the non-naturally occurring CKP that binds VEGF-Afurther comprises an L2 comprising the amino acid sequence KQDSD (SEQ IDNO: 93). In certain embodiments, the non-naturally occurring CKP thatbinds VEGF-A further comprises an L3 comprising the amino acid sequenceLAG. In certain embodiments, the non-naturally occurring CKP that bindsVEGF-A comprises an L4 comprising V or F. In certain embodiments, thenon-naturally occurring CKP that binds VEGF-A comprises an L5 comprisingthe amino acid sequence G/E/Y/Q/H-P/M/W/Y-N/Y/W-G/D/T/Q/R/S-F/A/E/S (SEQID NO: 20) or SWWPSL (SEQ ID NO: 237).

In certain embodiments, the non-naturally occurring CKP that bindsVEGF-A comprises an L1 comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 8-14 and 225-230, with reference toscaffold structure I above. In certain embodiments, the non-naturallyoccurring CKP that binds VEGF-A further comprises an L2 comprising theamino acid sequence KQDSD (SEQ ID NO: 93). In certain embodiments, thenon-naturally occurring CKP that binds VEGF-A further comprises an L3comprising the amino acid sequence LAG. In certain embodiments, thenon-naturally occurring CKP that binds VEGF-A comprises an L4 comprisingV or F. In certain embodiments, the non-naturally occurring CKP thatbinds VEGF-A further comprises an L5 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs 15-18 and 233-238). Theamino acid sequences of SEQ ID NOs 8-18, 225-230, and 233-238 areprovided in Table 1 below.

TABLE 1 ETDWYPHQID (SEQ ID NO: 225) GETVFEQFLW (SEQ ID NO: 226) HMMYDY(SEQ ID NO: 227) KKWQWWYM (SEQ ID NO: 228) PAIQNWKEHP (SEQ ID NO: 229)PTTRFKQY (SEQ ID NO: 8) QDPTFNWALY (SEQ ID NO: 9) QLMHPFWG(SEQ ID NO: 230) QLMQPFWG (SEQ ID NO: 10) RDLDVKWD (SEQ ID NO: 11)RTPWEPHDIT (SEQ ID NO: 12) TTPWPPHEIM (SEQ ID NO: 13) VTPWKPHWIN(SEQ ID NO: 14) GPNGF (SEQ ID NO: 233) GPNGF (SEQ ID NO: 234) EMYDA(SEQ ID NO: 235) YPWTE (SEQ ID NO: 236) SWWPSL (SEQ ID NO: 237) GPNGF(SEQ ID NO: 15) QMYQS (SEQ ID NO: 16) HWYRS (SEQ ID NO: 238) HWYQS(SEQ ID NO: 17) QYYSS (SEQ ID NO: 18) GPNGF (SEQ ID NO: 19)

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence on any one of SEQ ID NOs: 21-17 and239-244. The amino acid sequences of SEQ ID NOs: 21-17 and 239-244 areprovided in Table 2 below.

TABLE 2 GCETDWYPHQIDCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 239)GCGETVFEQFLWCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 240)GCHMMYDYCKQDSDCLAGCVCEMYDACG (SEQ ID NO: 241)GCKKWQWWYMCKQDSDCLAGCVCYPWTECG (SEQ ID NO: 242)GCPAIQNWKEHPCKQDSDCLAGCVCSWWPSLCG (SEQ ID NO: 243)GCPTTRFKQYCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 21)GCQDPTFNWALYCKQDSDCLAGCVCQMYQSCG (SEQ ID NO: 22)GCQLMHPFWGCKQDSDCLAGCVCHWYRSCG (SEQ ID NO: 244)GCQLMQPFWGCKQDSDCLAGCVCHWYQSCG (SEQ ID NO: 23)GCRDLDVKWDCKQDSDCLAGCFCQYYSSCG (SEQ ID NO: 24)GCRTPWEPHDITCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 25)GCTTPWPPHEIMCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 26)GCVTPWKPHWINCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 27)

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises an L1 comprising the amino acid sequenceQ/H/E/N/D-L/V/R/P/I-M/F/L-Q/E/R/L-P-F/A/L/S-W-G (SEQ ID NO: 358), withreference to scaffold structure I above. In certain embodiments thenon-naturally occurring VEGF-A binding CKP further comprises an L2comprising the amino acid sequence KQDSD (SEQ ID NO: 93). In certainembodiments the non-naturally occurring VEGF-A binding CKP furthercomprises an L3 comprising the amino acid sequence LAG. In certainembodiments the non-naturally occurring VEGF-A binding CKP furthercomprises an L4 comprising the amino acid V. IN certain embodiments thenon-naturally occurring VEGF-A binding CKP further comprises an L5comprising the amino acid sequence HWYQS (SEQ ID NO: 17).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises an L1 comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs 33, 36, and 245-253, with reference toscaffold structure I. In certain embodiments the non-naturally occurringVEGF-A binding CKP further comprises an L2 comprising the amino acidsequence KQDSD (SEQ ID NO: 93). In certain embodiments the non-naturallyoccurring VEGF-A binding CKP further comprises an L3 comprising theamino acid sequence LAG. In certain embodiments the non-naturallyoccurring VEGF-A binding CKP further comprises an L4 comprising theamino acid V. In certain embodiments the non-naturally occurring VEGF-Abinding CKP further comprises an L5 comprising the amino acid sequenceHWYQS (SEQ ID NO: 17). The amino acid sequences of SEQ ID NOs 33, 36,and 245-253 are provided in Table 3 below:

TABLE 3 HLFEPLWG (SEQ ID NO: 245) QVMRPFWG (SEQ ID NO: 246) QVMQPAWG(SEQ ID NO: 247) HRLQPLWG (SEQ ID NO: 248) ELLQPSWG (SEQ ID NO: 249)NPMLPFWG (SEQ ID NO: 368) NVLLPLWG (SEQ ID NO: 250) DIMQPLWG(SEQ ID NO: 36) DLMQPLWG (SEQ ID NO: 251) NPMLPLWG (SEQ ID NO: 252)QVLQPSWG (SEQ ID NO: 253)

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence on any one of SEQ ID NOs: 265-275. Theamino acid sequences of SEQ ID NOs: 265-275 are provided in Table 4below.

TABLE 4 GCHLFEPLWGCKQDSDCLAGCVCHWYQSCG (SEQ ID NO: 265)GCQVMRPFWGCKQDSDCLAGCVCHWYQSCG (SEQ ID NO: 266)GCQVMQPAWGCKQDSDCLAGCVCHWYQSCG (SEQ ID NO: 267)GCHRLQPLWGCKQDSDCLAGCVCHWYQSCG (SEQ ID NO: 268)GCELLQPSWGCKQDSDCLAGCVCHWYQSCG (SEQ ID NO: 269)GCNPMLPFWGCKQDSDCLAGCVCHWYQSCG (SEQ ID NO: 270)GCNVLLPLWGCKQDSDCLAGCVCHWYQSCG (SEQ ID NO: 271)GCDIMQPLWGCKQDSDCLAGCVCHWYQSCG (SEQ ID NO: 272)GCDLMQPLWGCKQDSDCLAGCVCHWYQSCG (SEQ ID NO: 273)GCNPMLPLWGCKQDSDCLAGCVCHWYQSCG (SEQ ID NO: 58)GCQVLQPSWGCKQDSDCLAGCVCHWYQSCG (SEQ ID NO: 275)

In certain embodiments, the non-naturally occurring VEGF-A binding CKPfurther comprises an L1 comprising the amino acid sequence QLMQPFWG (SEQID NO: 10), with reference to scaffold structure I. In certainembodiments the non-naturally occurring VEGF-A binding CKP furthercomprises an L2 comprising the amino acid sequence KQDSD (SEQ ID NO:93). In certain embodiments the non-naturally occurring VEGF-A bindingCKP further comprises an L3 comprising the amino acid sequence LAG. Incertain embodiments the non-naturally occurring VEGF-A binding CKPfurther comprises an L4 comprising the amino acid V. In certainembodiments, the non-naturally occurring VEGF-A binding CKP comprises anL5 comprising the amino acid sequence R/H-W-Y-N/Q/H-S(SEQ ID NO: 359).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPfurther comprises an L1 comprising the amino acid sequence QLMQPFWG (SEQID NO: 10), with reference to scaffold structure I above. In certainembodiments the non-naturally occurring VEGF-A binding CKP furthercomprises an L2 comprising the amino acid sequence KQDSD (SEQ ID NO:93). In certain embodiments the non-naturally occurring VEGF-A bindingCKP further comprises an L3 comprising the amino acid sequence LAG. Incertain embodiments the non-naturally occurring VEGF-A binding CKPfurther comprises an L4 comprising the amino acid V. In certainembodiments, the non-naturally occurring VEGF-A binding CKP comprises anL5 comprising an amino acid sequence selected from the group consistingof HWYQS (SEQ ID NO: 17), RWYHS (SEQ ID NO: 43), and RWYNS (SEQ ID NO:133).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 23, 276, and 278. SEQ ID NOs 23, 276, and 278 are providedin Table 5 below.

TABLE 5 GCQLMQPFWGCKQDSDCLAGCVCRWYNSCG (SEQ ID NO: 276)GCQLMQPFWGCKQDSDCLAGCVCHWYQSCG (SEQ ID NO: 23)GCQLMQPFWGCKQDSDCLAGCVCRWYHSCG (SEQ ID NO: 278)

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises an L1 comprising the amino acid sequenceQ/D/K/N/A/R/H-L/V/I/R/P/V/-M/L/S/T/F-Q/E/L/H-P-F/L/M/Y/S-W-G (SEQ ID NO:40), with reference to scaffold structure I. In certain embodiments thenon-naturally occurring VEGF-A binding CKP further comprises an L2comprising the amino acid sequence KQDSD (SEQ ID NO: 93). In certainembodiments the non-naturally occurring VEGF-A binding CKP furthercomprises an L3 comprising the amino acid sequence LAG. In certainembodiments the non-naturally occurring VEGF-A binding CKP furthercomprises an L4 comprising the amino acid V. In certain embodiments thenon-naturally occurring VEGF-A binding CKP further comprises an L5comprising the amino acid sequence H/L/R/Q/-W/F/Y-Y-Q/N/K/H/D/E-S(SEQ IDNO: 360).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises an L1 comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 28-39 and 254-261, with reference toscaffold structure I. In certain embodiments the non-naturally occurringVEGF-A binding CKP further comprises an L2 comprising the amino acidsequence KQDSD (SEQ ID NO: 93). In certain embodiments the non-naturallyoccurring VEGF-A binding CKP further comprises an L3 comprising theamino acid sequence LAG. In certain embodiments the non-naturallyoccurring VEGF-A binding CKP further comprises an L4 comprising theamino acid V. In certain embodiments the non-naturally occurring VEGF-Abinding CKP further comprises an L5 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 17, 41-46, 133,262-264, and 567. The amino acid sequences of SEQ ID NOs: 17, 28-39,41-46, 133, 254-264, and 567 are provided in Table 6 below:

TABLE 6 DVLQPFWG (SEQ ID NO: 28) HWYQS (SEQ ID NO: 17)QISQPFWG (SEQ ID NO: 29) HFYNS (SEQ ID NO: 41) DRMQPLWG (SEQ ID NO: 30)LWYKS (SEQ ID NO: 42) QLLEPMWG (SEQ ID NO: 254) HWYNS (SEQ ID NO: 46)KLLQPMWG (SEQ ID NO: 255) QWYKS (SEQ ID NO: 262)DRMQPYWG (SEQ ID NO: 256) RWYHS (SEQ ID NO: 43) NLMLPFWG (SEQ ID NO: 31)RWYQS (SEQ ID NO: 44) QRTQPFWG (SEQ ID NO: 32) LWYDS (SEQ ID NO: 263)KIMQPLWG (SEQ ID NO: 257) QYYQS (SEQ ID NO: 45)NLMHPFWG (SEQ ID NO: 258) RWYNS (SEQ ID NO: 133)NIMLPFWG (SEQ ID NO: 33) QWYQS (SEQ ID NO: 264) DPMQPFWG (SEQ ID NO: 34)NPMLPLWG (SEQ ID NO: 38) DVMQPYWG (SEQ ID NO: 35)KLFEPLWG (SEQ ID NO: 39) DIMQPLWG (SEQ ID NO: 36) RWYES (SEQ ID NO: 567)ALLQPLWG (SEQ ID NO: 259) QLLQPLWG (SEQ ID NO: 37)RLLEPSWG (SEQ ID NO: 260) HLLLPLWG (SEQ ID NO: 261)

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 47-59 and 279-286. The amino acid sequences of SEQ ID NOs:47-59 and 279-286 are provided in Table 7 below.

TABLE 7 GCDVLQPFWGCKQDSDCLAGCVCHWYQSCG (SEQ ID NO: 47)GCQISQPFWGCKQDSDCLAGCVCHFYNSCG (SEQ ID NO: 48)GCDRMQPLWGCKQDSDCLAGCVCLWYKSCG (SEQ ID NO: 49)GCQLLEPMWGCKQDSDCLAGCVCHWYNSCG (SEQ ID NO: 279)GCKLLQPMWGCKQDSDCLAGCVCRWYQSCG (SEQ ID NO: 280)GCDRMQPYWGCKQDSDCLAGCVCQWYKSCG (SEQ ID NO: 281)GCNLMLPFWGCKQDSDCLAGCVCRWYHSCG (SEQ ID NO: 50)GCQRTQPFWGCKQDSDCLAGCVCRWYQSCG (SEQ ID NO: 51)GCKIMQPLWGCKQDSDCLAGCVCLWYDSCG (SEQ ID NO: 282)GCNLMHPFWGCKQDSDCLAGCVCHWYQSCG (SEQ ID NO: 283)GCNIMLPFWGCKQDSDCLAGCVCQYYQSCG (SEQ ID NO: 52)GCNPMLPFWGCKQDSDCLAGCVCHWYQSCG (SEQ ID NO: 53)GCDPMQPFWGCKQDSDCLAGCVCRWYQSCG (SEQ ID NO: 54)GCDVMQPYWGCKQDSDCLAGCVCHWYNSCG (SEQ ID NO: 55)GCDIMQPLWGCKQDSDCLAGCVCHWYQSCG (SEQ ID NO: 56)GCALLQPLWGCKQDSDCLAGCVCRWYNSCG (SEQ ID NO: 284)GCQLLQPLWGCKQDSDCLAGCVCRWYQSCG (SEQ ID NO: 57)GCRLLEPSWGCKQDSDCLAGCVCQWYQSCG (SEQ ID NO: 285)GCHLLLPLWGCKQDSDCLAGCVCRWYHSCG (SEQ ID NO: 286)GCNPMLPLWGCKQDSDCLAGCVCHWYQSCG (SEQ ID NO: 58)GCKLFEPLWGCKQDSDCLAGCVCRWYESCG (SEQ ID NO: 59)

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises an L1 comprising the amino acid sequenceQ/D/K/W/E/L/R-D/N-P/R/L/T-T/S/L/K-F/V/L/I-N/D-W-A/S/G-L/V/E/T/Q/D-F/Y(SEQID NO: 70), with reference to scaffold structure I. In certainembodiments the non-naturally occurring VEGF-A binding CKP furthercomprises an L2 comprising the amino acid sequence KQDSD (SEQ ID NO:93). In certain embodiments the non-naturally occurring VEGF-A bindingCKP further comprises an L3 comprising the amino acid sequence LAG. Incertain embodiments the non-naturally occurring VEGF-A binding CKPfurther comprises an L4 comprising the amino acid V. In certainembodiments the non-naturally occurring VEGF-A binding CKP furthercomprises an L5 comprising the amino acid sequenceQ/R-M/L/F/H-Y/F/H-D/Q/N/K-S/T (SEQ ID NO: 80).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises an L1 comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 60-69 and 287-291. In certainembodiments the non-naturally occurring VEGF-A binding CKP furthercomprises an L2 comprising the amino acid sequence KQDSD (SEQ ID NO:93). In certain embodiments the non-naturally occurring VEGF-A bindingCKP further comprises an L3 comprising the amino acid sequence LAG. Incertain embodiments the non-naturally occurring VEGF-A binding CKPfurther comprises an L4 comprising the amino acid V. In certainembodiments the non-naturally occurring VEGF-A binding CKP furthercomprises an L5 comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 16, 71-79, 274, and 292. The amino acidsequences of SEQ ID NOs: 16, 60-69, 71-79, 274, and 287-292 are providedin Table 8 below.

TABLE 8 DDPSFDWSVY (SEQ ID NO: 287) RMYDS (SEQ ID NO: 292)KNPLFNWALY (SEQ ID NO: 60) QLFDS (SEQ ID NO: 71)QDPTVNWAVY (SEQ ID NO: 61) QFYQS (SEQ ID NO: 72)QDPTFNWAEY (SEQ ID NO: 62) QLYQS (SEQ ID NO: 73)WDPTFNWALY (SEQ ID NO: 288) QMYDS (SEQ ID NO: 76)QDPTLNWATY (SEQ ID NO: 289) QMYQS (SEQ ID NO: 16)EDPTVDWAQY (SEQ ID NO: 290) QMHQS (SEQ ID NO: 74)QDPSLNWADY (SEQ ID NO: 63) QMYNS (SEQ ID NO: 75)LDRTLNWALY (SEQ ID NO: 64) QLYQS (SEQ ID NO: 73)LDPSFNWSLY (SEQ ID NO: 65) QHYKT (SEQ ID NO: 77)RDLTINWALF (SEQ ID NO: 66) QLFNS (SEQ ID NO: 78)KDTTFNWGLF (SEQ ID NO: 291) QLYNS (SEQ ID NO: 79)LDPTVNWALF (SEQ ID NO: 67) QMFNS (SEQ ID NO: 274)QDPKLNWAVY (SEQ ID NO: 68) LDPSFDWALY (SEQ ID NO: 69)

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 81-91 and 293-297. The amino acid sequences of SEQ ID NOs:81-91 and 293-297 are provided in Table 9 below.

TABLE 9 GCDDPSFDWSVYCKQDSDCLAGCVCRMYDSCG (SEQ ID NO: 293)GCKNPLFNWALYCKQDSDCLAGCVCQLFDSCG (SEQ ID NO: 81)GCQDPTVNWAVYCKQDSDCLAGCVCQFYQSCG (SEQ ID NO: 82)GCQDPTFNWAEYCKQDSDCLAGCVCQLYQSCG (SEQ ID NO: 83)GCWDPTFNWALYCKQDSDCLAGCVCQMYDSCG (SEQ ID NO: 294)GCQDPTFNWAEYCKQDSDCLAGCVCQMYQSCG (SEQ ID NO: 84)GCQDPSLNWADYCKQDSDCLAGCVCQMHQSCG (SEQ ID NO: 85)GCQDPTLNWATYCKQDSDCLAGCVCQMYQSCG (SEQ ID NO: 295)GCEDPTVDWAQYCKQDSDCLAGCVCQMYQSCG (SEQ ID NO: 296)GCLDRTLNWALYCKQDSDCLAGCVCQMYNSCG (SEQ ID NO: 86)GCLDPSFNWSLYCKQDSDCLAGCVCQMYDSCG (SEQ ID NO: 87)GCRDLTINWALFCKQDSDCLAGCVCQMFNSCG (SEQ ID NO: 88)GCKDTTFNWGLFCKQDSDCLAGCVCQLYQSCG (SEQ ID NO: 297)GCLDPTVNWALFCKQDSDCLAGCVCQHYKTCG (SEQ ID NO: 89)GCQDPKLNWAVYCKQDSDCLAGCVCQLFNSCG (SEQ ID NO: 90)GCLDPSFDWALYCKQDSDCLAGCVCQLYNSCG (SEQ ID NO: 91)

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises an L1 comprising the amino acid sequence NIMLPFWG (SEQ ID NO:33), with reference to scaffold structure I. In certain embodiments, thenon-naturally occurring VEGF-A binding CKP further comprises an L2comprising the amino acid sequenceK/G/Q/S/N-Q/L/P/A/V/T/R/W/K/G/Y-D/S/E/N-S/F/Y/L/F/Q/M-D/E/N/A/L/F/H/Q(SEQ ID NO: 98). In certain embodiments the non-naturally occurringVEGF-A binding CKP further comprises an L3 comprising the amino acidsequence LAG. In certain embodiments the non-naturally occurring VEGF-Abinding CKP further comprises an L4 comprising the amino acid V. Incertain embodiments the non-naturally occurring VEGF-A binding CKPfurther comprises an L5 comprising the amino acid sequence QYYQS (SEQ IDNO: 45).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises an L1 comprising the amino acid sequence NIMLPFWG (SEQ ID NO:33), with reference to scaffold structure I. In certain embodiments, thenon-naturally occurring VEGF-A binding CKP further comprises an L2comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 94-97 and 298-309. In certain embodiments the non-naturallyoccurring VEGF-A binding CKP further comprises an L3 comprising theamino acid sequence LAG. In certain embodiments the non-naturallyoccurring VEGF-A binding CKP further comprises an L4 comprising theamino acid V. In certain embodiments the non-naturally occurring VEGF-Abinding CKP further comprises an L5 comprising the amino acid sequenceQYYQS (SEQ ID NO: 45). The amino acid sequences of SEQ ID NOs: 94-97 and298-309 are provided in Table 10 below.

TABLE 10 GQSFE (SEQ ID NO: 94) GWDQF (SEQ ID NO: 304)GLDYD (SEQ ID NO: 95) GKDFH (SEQ ID NO: 305) GPELN (SEQ ID NO: 298)GPDLQ (SEQ ID NO: 96) QADYA (SEQ ID NO: 299) SGDFA (SEQ ID NO: 306)GVDYL (SEQ ID NO: 300) GKELN (SEQ ID NO: 307) GTNFL (SEQ ID NO: 301)GWSMD (SEQ ID NO: 308) SRDFD (SEQ ID NO: 302) GYDLQ (SEQ ID NO: 309)NRDFL (SEQ ID NO: 303) GRDFE (SEQ ID NO: 97)

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 99-102 and 310-321. The amino acid sequences of SEQ ID NOs:99-102 and 310-321 are provided in Table 11 below.

TABLE 11 GCNIMLPFWGCGQSFECLAGCVCQYYQSCG (SEQ ID NO: 99)GCNIMLPFWGCGLDYDCLAGCVCQYYQSCG (SEQ ID NO: 100)GCNIMLPFWGCGPELNCLAGCVCQYYQSCG (SEQ ID NO: 310)GCNIMLPFWGCQADYACLAGCVCQYYQSCG (SEQ ID NO: 311)GCNIMLPFWGCGVDYLCLAGCVCQYYQSCG (SEQ ID NO: 312)GCNIMLPFWGCGTNFLCLAGCVCQYYQSCG (SEQ ID NO: 313)GCNIMLPFWGCSRDFDCLAGCVCQYYQSCG (SEQ ID NO: 314)GCNIMLPFWGCNRDFLCLAGCVCQYYQSCG (SEQ ID NO: 315)GCNIMLPFWGCGWDQFCLAGCVCQYYQSCG (SEQ ID NO: 316)GCNIMLPFWGCGKDFHCLAGCVCQYYQSCG (SEQ ID NO: 317)GCNIMLPFWGCGPDLQCLAGCVCQYYQSCG (SEQ ID NO: 101)GCNIMLPFWGCSGDFACLAGCVCQYYQSCG (SEQ ID NO: 318)GCNIMLPFWGCGKELNCLAGCVCQYYQSCG (SEQ ID NO: 319)GCNIMLPFWGCGWSMDCLAGCVCQYYQSCG (SEQ ID NO: 320)GCNIMLPFWGCGYDLQCLAGCVCQYYQSCG (SEQ ID NO: 321)GCNIMLPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 102)

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises an L1 comprising the amino acid sequenceN-I-M/L-L/S/T/Q/N/E/D-P-F/Y/S-WG (SEQ ID NO: 454), with reference toscaffold structure I. In certain embodiments, the non-naturallyoccurring VEGF-A binding CKP comprises an L1 comprising the amino acidsequence NIMLPFWG (SEQ ID NO: 33), with reference to scaffold structureI. In certain embodiments, the non-naturally occurring VEGF-A bindingCKP further comprises an L2 comprising the amino acid sequence GRDFE(SEQ ID NO: 97). In certain embodiments the non-naturally occurringVEGF-A binding CKP further comprises an L3 comprising the amino acidsequence L/V/M/F-A/Q/E/S/N/Y/I/T-G/Q/R/D/T/N/E. In certain embodimentsthe non-naturally occurring VEGF-A binding CKP further comprises an L4comprising the amino acid V or I. In certain embodiments thenon-naturally occurring VEGF-A binding CKP further comprises an L5comprising the amino acid sequence QYYQS (SEQ ID NO: 45).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises an L1 comprising the amino acid sequence selected from thegroup consisting of NIMLPFWG (SEQ ID NO: 33), NILLPFWG (SEQ ID NO: 396),NILLPYWG (SEQ ID NO: 397), NIMSPFWG (SEQ ID NO: 398), NIMTPFWG (SEQ IDNO: 399), NIMQPFWG (SEQ ID NO: 400), NIMNPFWG (SEQ ID NO: 401), NIMEPFWG(SEQ ID NO: 402), NIMDPFWG (SEQ ID NO: 403), NIMLPSWG (SEQ ID NO: 414),and NIMLPYWG (SEQ ID NO: 141) with reference to scaffold structure I. Incertain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises an L1 comprising the amino acid sequence NIMLPFWG (SEQ ID NO:33), with reference to scaffold structure V or I. In certainembodiments, the non-naturally occurring VEGF-A binding CKP furthercomprises an L2 comprising the amino acid sequence GRDFE (SEQ ID NO:97). In certain embodiments the non-naturally occurring VEGF-A bindingCKP further comprises an L3 comprising an amino acid sequence selectedfrom the group consisting of: LQQ, VER, MSD, MNQ, MQT, VYQ, FIN, VSQ,VTE, FYE, MEQ, and VYR. In certain embodiments the non-naturallyoccurring VEGF-A binding CKP further comprises an L4 comprising theamino acid I. In certain embodiments the non-naturally occurring VEGF-Abinding CKP further comprises an L5 comprising the amino acid sequenceQYYQS (SEQ ID NO: 45).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 103-114. The amino acid sequences of SEQ ID NOs: 103-114 areprovided in Table 12 below.

TABLE 12 GCNIMLPFWGCGRDFECLQQCICQYYQSCG (SEQ ID NO: 103)GCNIMLPFWGCGRDFECVERCICQYYQSCG (SEQ ID NO: 104)GCNIMLPFWGCGRDFECMSDCICQYYQSCG (SEQ ID NO: 105)GCNIMLPFWGCGRDFECMNQCICQYYQSCG (SEQ ID NO: 106)GCNIMLPFWGCGRDFECMQTCICQYYQSCG (SEQ ID NO: 107)GCNIMLPFWGCGRDFECVYQCICQYYQSCG (SEQ ID NO: 108)GCNIMLPFWGCGRDFECFINCICQYYQSCG (SEQ ID NO: 109)GCNIMLPFWGCGRDFECVSQCICQYYQSCG (SEQ ID NO: 110)GCNIMLPFWGCGRDFECVTECICQYYQSCG (SEQ ID NO: 111)GCNIMLPFWGCGRDFECFYECICQYYQSCG (SEQ ID NO: 112)GCNIMLPFWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 113)GCNIMLPFWGCGRDFECVYRCICQYYQSCG (SEQ ID NO: 114)GCDVLQPYWGCGPDIDCLSNCICHWYNSCG (SEQ ID NO: 386)GCNILLPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 405)GCNILLPYWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 406)GCNIMSPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 407)GCNIMTPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 408)GCNIMQPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 409)GCNIMNPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 410)GCNIMEPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 411)GCNIMDPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 412)GCNIMLPSWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 415)GCNIMLPFWGCGRDFECLSGCVCQYYQSCG (SEQ ID NO: 421)GCNIMLPFWGCGRDFECLTGCVCQYYQSCG (SEQ ID NO: 422)GCNIMLPFWGCGRDFECLEGCVCQYYQSCG (SEQ ID NO: 423)GCNIMLPYWGCGRDFECLAGCLCQYYQSCG (SEQ ID NO: 424)GCNIMLPYWGCGRDFECLAGCICQYYQSCG (SEQ ID NO: 425)GCNIMLPYWGCGRDFECLAGCVCQYYQSCS (SEQ ID NO: 431)GCNILLPYWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 435)

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises an L1 comprising the amino acid sequence DVMQPYWG (SEQ ID NO:35), with reference to scaffold structure I. In certain embodiments, thenon-naturally occurring VEGF-A binding CKP further comprises an L2comprising the amino acid sequenceK/G/D/A/E-Q/E/R/V/P/D/M/G/N/L/A/F-D/N/Y/S-S/F/L/I/M/Y/V/N/E-D/L/Q/S/E/T/L/A/N(SEQ ID NO: 121). In certain embodiments the non-naturally occurringVEGF-A binding CKP further comprises an L3 comprising the amino acidsequence LAG. In certain embodiments the non-naturally occurring VEGF-Abinding CKP further comprises an L4 comprising the amino acid V. Incertain embodiments the non-naturally occurring VEGF-A binding CKPfurther comprises an L5 comprising the amino acid sequence HWYNS (SEQ IDNO: 46).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises an L1 comprising the amino acid sequence DVMQPYWG (SEQ ID NO:35), with reference to scaffold structure I. In certain embodiments, thenon-naturally occurring VEGF-A binding CKP further comprises an L2comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 117-120, 211, and 322-339. In certain embodiments thenon-naturally occurring VEGF-A binding CKP further comprises an L3comprising the amino acid sequence LAG. In certain embodiments thenon-naturally occurring VEGF-A binding CKP further comprises an L4comprising the amino acid V. In certain embodiments the non-naturallyoccurring VEGF-A binding CKP further comprises an L5 comprising theamino acid sequence HWYNS (SEQ ID NO: 46). The amino acid sequences ofSEQ ID NOs: 117-120, 211, and 322-339 are provided in Table 13 below.

TABLE 13 GENFL (SEQ ID NO: 117) DGDFD (SEQ ID NO: 331)GRDLQ (SEQ ID NO: 322) AGDFE (SEQ ID NO: 332) GVDLS (SEQ ID NO: 323)EMDFD (SEQ ID NO: 120) GPDID (SEQ ID NO: 118) GNSFE (SEQ ID NO: 333)GDDLE (SEQ ID NO: 324) GQDLT (SEQ ID NO: 334) GVDMT (SEQ ID NO: 325)GENLA (SEQ ID NO: 335) GMDIE (SEQ ID NO: 326) GQDYN (SEQ ID NO: 336)DGDYQ (SEQ ID NO: 327) GADLS (SEQ ID NO: 337) GNDVS (SEQ ID NO: 328)GFDMD (SEQ ID NO: 338) GRDMD (SEQ ID NO: 119) GESLS (SEQ ID NO: 211)AGDEL (SEQ ID NO: 329) DLNYE (SEQ ID NO: 339) GLDEE (SEQ ID NO: 330)

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 122-126 and 340-357. The amino acid sequences of SEQ ID NOs:SEQ ID NOs: 122-126 and 340-357 are provided in Table 14 below.

TABLE 14 GCDVMQPYWGCGENFLCLAGCVCHWYNSCG (SEQ ID NO: 122)GCDVMQPYWGCGRDLQCLAGCVCHWYNSCG (SEQ ID NO: 340)GCDVMQPYWGCGVDLSCLAGCVCHWYNSCG (SEQ ID NO: 341)GCDVMQPYWGCGPDIDCLAGCVCHWYNSCG (SEQ ID NO: 123)GCDVMQPYWGCGDDLECLAGCVCHWYNSCG (SEQ ID NO: 342)GCDVMQPYWGCGVDMTCLAGCVCHWYNSCG (SEQ ID NO: 343)GCDVMQPYWGCGMDIECLAGCVCHWYNSCG (SEQ ID NO: 344)GCDVMQPYWGCDGDYQCLAGCVCHWYNSCG (SEQ ID NO: 345)GCDVMQPYWGCGNDVSCLAGCVCHWYNSCG (SEQ ID NO: 346)GCDVMQPYWGCGRDMDCLAGCVCHWYNSCG (SEQ ID NO: 124)GCDVMQPYWGCAGDELCLAGCVCHWYNSCG (SEQ ID NO: 347)GCDVMQPYWGCGLDEECLAGCVCHWYNSCG (SEQ ID NO: 348)GCDVMQPYWGCDGDFDCLAGCVCHWYNSCG (SEQ ID NO: 349)GCDVMQPYWGCAGDFECLAGCVCHWYNSCG (SEQ ID NO: 350)GCDVMQPYWGCEMDFDCLAGCVCHWYNSCG (SEQ ID NO: 125)GCDVMQPYWGCGNSFECLAGCVCHWYNSCG (SEQ ID NO: 351)GCDVMQPYWGCGQDLTCLAGCVCHWYNSCG (SEQ ID NO: 352)GCDVMQPYWGCGENLACLAGCVCHWYNSCG (SEQ ID NO: 353)GCDVMQPYWGCGQDYNCLAGCVCHWYNSCG (SEQ ID NO: 354)GCDVMQPYWGCGADLSCLAGCVCHWYNSCG (SEQ ID NO: 355)GCDVMQPYWGCGFDMDCLAGCVCHWYNSCG (SEQ ID NO: 356)GCDVMQPYWGCGESLSCLAGCVCHWYNSCG (SEQ ID NO: 126)GCDVMQPYWGCDLNYECLAGCVCHWYNSCG (SEQ ID NO: 357)

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises an L1 comprising the amino acid sequenceD-V-M/L-Q/K/D-P-Y/M/T/L-W-G (SEQ ID NO: 130), with reference to scaffoldstructure I. In certain embodiments, the non-naturally occurring VEGF-Abinding CKP further comprises an L2 comprising the amino acid sequenceKQDSD (SEQ ID NO: 93). In certain embodiments the non-naturallyoccurring VEGF-A binding CKP further comprises an L3 comprising theamino acid sequence LAG. In certain embodiments the non-naturallyoccurring VEGF-A binding CKP further comprises an L4 comprising theamino acid V. In certain embodiments the non-naturally occurring VEGF-Abinding CKP further comprises an L5 comprising the amino acid sequenceH/L/Q/R-W-Y-N-S (SEQ ID NO: 134).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises an L1 comprising an amino acid sequence selected from SEQ IDNOs: 127-129, with reference to scaffold structure I. In certainembodiments, the non-naturally occurring VEGF-A binding CKP furthercomprises an L2 comprising the amino acid sequence KQDSD (SEQ ID NO:93). In certain embodiments the non-naturally occurring VEGF-A bindingCKP further comprises an L3 comprising the amino acid sequence LAG. Incertain embodiments the non-naturally occurring VEGF-A binding CKPfurther comprises an L4 comprising the amino acid V. In certainembodiments the non-naturally occurring VEGF-A binding CKP furthercomprises an L5 comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 131-133. The amino acid sequences of SEQID NOs: 127-129 and 131-133 are provided in Table 15 below:

TABLE 15 DVMKPMWG (SEQ ID NO: 127) QWYNS (SEQ ID NO: 131)DVLDPTWG (SEQ ID NO: 128) LWYNS (SEQ ID NO: 132)DVLQPLWG (SEQ ID NO: 129) RWYNS (SEQ ID NO: 133)

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 135-137. The amino acid sequences of SEQ ID NOs: 135-127 areprovided in Table 16 below:

TABLE 16 GCDVMKPMWGCKQDSDCLAGCVCQWYNSCG (SEQ ID NO: 135)GCDVLDPTWGCKQDSDCLAGCVCLWYNSCG (SEQ ID NO: 136)GCDVLQPLWGCKQDSDCLAGCVCRWYNSCG (SEQ ID NO: 137)

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises an L1 comprising the amino acid sequence DVMQPYWG (SEQ ID NO:35), with reference to scaffold structure I. In certain embodiments, thenon-naturally occurring VEGF-A binding CKP further comprises an L2comprising the amino acid sequence GPDID (SEQ ID NO: 118). In certainembodiments the non-naturally occurring VEGF-A binding CKP furthercomprises an L3 comprising the amino acid sequence L/F-A/V/S-G/R/N. Incertain embodiments the non-naturally occurring VEGF-A binding CKPfurther comprises an L4 comprising an amino acid selected from V, I, andL. In certain embodiments the non-naturally occurring VEGF-A binding CKPfurther comprises an L5 comprising the amino acid sequence HWYNS (SEQ IDNO: 46).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises an L1 comprising the amino acid sequence DVMQPYWG (SEQ ID NO:35), with reference to scaffold structure I. In certain embodiments, thenon-naturally occurring VEGF-A binding CKP further comprises an L2comprising the amino acid sequence GPDID (SEQ ID NO: 118). In certainembodiments the non-naturally occurring VEGF-A binding CKP furthercomprises an L3 comprising an amino acid sequence FVR and LSN. Incertain embodiments the non-naturally occurring VEGF-A binding CKPfurther comprises an L4 comprising an amino acid selected from V, I, andL. In certain embodiments the non-naturally occurring VEGF-A binding CKPfurther comprises an L5 comprising the amino acid sequence HWYNS (SEQ IDNO: 46).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCDVMQPYWGCGPDIDCFVRCLCHWYNSCG (SEQ IDNO: 139). In certain embodiments, the non-naturally occurring VEGF-Abinding CKP comprises the amino acid sequenceGCDVMQPYWGCGPDIDCLSNCICHWYNSCG (SEQ ID NO: 140).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises an L1, L2, L3, L4 and/or L5 of any one of the non-naturallyoccurring VEGF-A binding CKPs disclosed herein. Thus, in certainembodiments, the non-naturally occurring VEGF-A binding CKP comprises anL1 comprising an amino acid sequence selected from the group consistingof SEQ ID NOs: 8-14, 28-39, 60-69, 127-129, 141, 225-230, 245-261,287-291, 396-403, and 414 with reference to scaffold structure I. Incertain embodiments, the non-naturally occurring VEGF-A binding CKPfurther comprises an L2 comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 93-97, 117-120, 211, 298-309, and322-339. In certain embodiments, the non-naturally occurring VEGF-Abinding CKP further comprises an L3 comprising an amino acid sequenceselected from the group consisting of LAG, LQQ, VER, MSD, MNQ, MQT, VYQ,FIN, VSQ, VTE, FYE, MEQ, and VYR, FVR and LSN. In certain embodiments,the non-naturally occurring VEGF-A binding CKP further comprises an L4comprising the amino acid V, F, I, or L. In certain embodiments, thenon-naturally occurring VEGF-A binding CKP further comprises an L5comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 15-18, 41-46, 71-79, 131-133, 233-238, 262-264, and 292. Incertain embodiments, the C-terminus of the non-naturally occurringcystine knot peptide (CKP) that binds to VEGF-A is modified (such ascapped). In certain embodiments, the N-terminus of the non-naturallyoccurring cystine knot peptide (CKP) that binds to VEGF-A is modified(such as capped). In certain embodiments, both the C- and N-termini ofthe non-naturally occurring cystine knot peptide (CKP) that binds toVEGF-A are modified (such as capped). In certain embodiments, theC-terminal carboxyl group of the non-naturally occurring cystine knotpeptide (CKP) that binds to VEGF-A is amidated. In certain embodiments,the N-terminal amine of the non-naturally occurring cystine knot peptide(CKP) that binds to VEGF-A is acetylated. In certain embodiments, theC-terminal carboxyl group of the non-naturally occurring cystine knotpeptide (CKP) that binds to VEGF-A is amidated and the N-terminal amineof the non-naturally occurring cystine knot peptide (CKP) that binds toVEGF-A is acetylated.

In certain embodiments, at least one amino acid is deleted from a VEGF-Abinding CKP provided herein. In certain embodiments, at least one aminoacid is deleted from the N-terminus. In certain embodiments, at leastone amino acid is deleted from the C-terminus. In certain embodiments,at least one amino acid is deleted from the N-terminus and theC-terminus. In certain embodiments, at least one internal amino acid isdeleted. In certain embodiments, the non-naturally occurring VEGF-Abinding CKP comprises the amino acid sequenceCNIMLPYWGCGRDFECLAGCVCQYYQSC (SEQ ID NO: 217). In certain embodiments,the C-terminus of the non-naturally occurring cystine knot peptide (CKP)that binds to VEGF-A is modified (such as capped). In certainembodiments, the N-terminus of the non-naturally occurring cystine knotpeptide (CKP) that binds to VEGF-A is modified (such as capped). Incertain embodiments, both the C- and N-termini of the non-naturallyoccurring cystine knot peptide (CKP) that binds to VEGF-A are modified(such as capped). In certain embodiments, the C-terminal carboxyl groupof the non-naturally occurring cystine knot peptide (CKP) that binds toVEGF-A is amidated. In certain embodiments, the N-terminal amine of thenon-naturally occurring cystine knot peptide (CKP) that binds to VEGF-Ais acetylated. In certain embodiments, the C-terminal carboxyl group ofthe non-naturally occurring cystine knot peptide (CKP) that binds toVEGF-A is amidated and the N-terminal amine of the non-naturallyoccurring cystine knot peptide (CKP) that binds to VEGF-A is acetylated.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises at least one amino acid addition. In certain embodiments, atleast one amino acid is added to the N-terminus. In certain embodiments,at least one amino acid is added to the C-terminus. In certainembodiments, at least one amino acid is added to the N-terminus and theC-terminus.

In certain embodiments, two amino acids are added to the N-terminus of anon-naturally occurring VEGF-A binding CKP provided herein. In certainembodiments, two amino acids are added to the N-terminus of thenon-naturally occurring VEGF-A binding CKP set forth inGCNIMLPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 102). In certain embodiments,the two amino acids added to the N-terminus of SEQ ID NO: 102 areF/I/G/T/V/L-H/A/S/R. In certain embodiments, the two amino acids addedto the N-terminus of SEQ ID NO: 102 are selected from the groupconsisting of: FH, IA, GS, TR, VH, and LS. In certain embodiments, thenon-naturally occurring VEGF-A binding CKP comprises an amino acidsequence set forth in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGFH (SEQ ID NO: 379).In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises an amino acid sequence set forth inGCNIMLPFWGCGRDFECLAGCVCQYYQSCGIA (SEQ ID NO: 380). In certainembodiments, the non-naturally occurring VEGF-A binding CKP comprises anamino acid sequence set forth in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGGS (SEQID NO: 381). In certain embodiments, the non-naturally occurring VEGF-Abinding CKP comprises an amino acid sequence set forth inGCNIMLPFWGCGRDFECLAGCVCQYYQSCGTR (SEQ ID NO: 382). In certainembodiments, the non-naturally occurring VEGF-A binding CKP comprises anamino acid sequence set forth in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGVH (SEQID NO: 383). In certain embodiments, the non-naturally occurring VEGF-Abinding CKP comprises an amino acid sequence set forth inGCNIMLPFWGCGRDFECLAGCVCQYYQSCGLS (SEQ ID NO: 384).

In certain embodiments, two amino acids are added to the N-terminus ofthe non-naturally occurring VEGF-A binding CKP set forth inGCDVLQPYWGCGPDIDCLSNCICHWYNSCG (SEQ ID NO: 386). In certain embodiments,the two amino acids added to the N-terminus of SEQ ID NO: 102 areR/W/P/D/Q/E/S-T/K/E/F/Q/L/S. In certain embodiments, the two amino acidsadded to the N-terminus of SEQ ID NO: 102 are selected from the groupconsisting of: RT, WK, PL, DE, QF, EQ, PT, RL, and SL. In certainembodiments, the non-naturally occurring VEGF-A binding CKP comprises anamino acid sequence set forth in GCDVLQPYWGCGPDIDCLSNCICHWYNSCGRT (SEQID NO: 387). In certain embodiments, the non-naturally occurring VEGF-Abinding CKP comprises an amino acid sequence set forth inGCDVLQPYWGCGPDIDCLSNCICHWYNSCGWK (SEQ ID NO: 388). In certainembodiments, the non-naturally occurring VEGF-A binding CKP comprises anamino acid sequence set forth in GCDVLQPYWGCGPDIDCLSNCICHWYNSCGPL (SEQID NO: 389). In certain embodiments, the non-naturally occurring VEGF-Abinding CKP comprises an amino acid sequence set forth inGCDVLQPYWGCGPDIDCLSNCICHWYNSCGDE (SEQ ID NO: 390). In certainembodiments, the non-naturally occurring VEGF-A binding CKP comprises anamino acid sequence set forth in GCDVLQPYWGCGPDIDCLSNCICHWYNSCGQF (SEQID NO: 391). In certain embodiments, the non-naturally occurring VEGF-Abinding CKP comprises an amino acid sequence set forth inGCDVLQPYWGCGPDIDCLSNCICHWYNSCGEQ (SEQ ID NO: 392). In certainembodiments, the non-naturally occurring VEGF-A binding CKP comprises anamino acid sequence set forth in GCDVLQPYWGCGPDIDCLSNCICHWYNSCGPT (SEQID NO: 393). In certain embodiments, the non-naturally occurring VEGF-Abinding CKP comprises an amino acid sequence set forth inGCDVLQPYWGCGPDIDCLSNCICHWYNSCGRL (SEQ ID NO: 394). In certainembodiments, the non-naturally occurring VEGF-A binding CKP comprises anamino acid sequence set forth in GCDVLQPYWGCGPDIDCLSNCICHWYNSCGSL (SEQID NO: 395).

In certain embodiments, three amino acids are added to the N-terminus ofa non-naturally occurring VEGF-A binding CKP provided herein. In certainembodiments, two amino acids are added to the N-terminus of thenon-naturally occurring VEGF-A binding CKP set forth inGCNIMLPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 102). In certain embodiments,the three amino acids added to the N-terminus of SEQ ID NO: 102 areP/N/T/D/E/Y/W-L/Y/F/H/D/P-I/Q/V/K/S/Y/H. In certain embodiments, thethree amino acids added to the N-terminus of SEQ ID NO: 102 are selectedfrom the group consisting of: PLI, NYQ, PLQ, TFQ, DLV, EHK, YLS, WDY,WPH, and PHQ. In certain embodiments, the non-naturally occurring VEGF-Abinding CKP comprises an amino acid sequence set forth inGCNIMLPFWGCGRDFECLAGCVCQYYQSCGPLI (SEQ ID NO: 369). In certainembodiments, the non-naturally occurring VEGF-A binding CKP comprises anamino acid sequence set forth in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGNYQ (SEQID NO: 370). In certain embodiments, the non-naturally occurring VEGF-Abinding CKP comprises an amino acid sequence set forth inGCNIMLPFWGCGRDFECLAGCVCQYYQSCGPLQ (SEQ ID NO: 371). In certainembodiments, the non-naturally occurring VEGF-A binding CKP comprises anamino acid sequence set forth in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGTFQ (SEQID NO: 372). In certain embodiments, the non-naturally occurring VEGF-Abinding CKP comprises an amino acid sequence set forth inGCNIMLPFWGCGRDFECLAGCVCQYYQSCGDLV (SEQ ID NO: 373). In certainembodiments, the non-naturally occurring VEGF-A binding CKP comprises anamino acid sequence set forth in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGEHK (SEQID NO: 374). In certain embodiments, the non-naturally occurring VEGF-Abinding CKP comprises an amino acid sequence set forth inGCNIMLPFWGCGRDFECLAGCVCQYYQSCGYLS (SEQ ID NO: 375). In certainembodiments, the non-naturally occurring VEGF-A binding CKP comprises anamino acid sequence set forth in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGWDY (SEQID NO: 376). In certain embodiments, the non-naturally occurring VEGF-Abinding CKP comprises an amino acid sequence set forth inGCNIMLPFWGCGRDFECLAGCVCQYYQSCGWPH (SEQ ID NO: 377). In certainembodiments, the non-naturally occurring VEGF-A binding CKP comprises anamino acid sequence set forth in GCNIMLPFWGCGRDFECLAGCVCQYYQSCGPHQ (SEQID NO: 378).

In certain embodiments, the C-terminus of the non-naturally occurringcystine knot peptide (CKP) that binds to VEGF-A is modified (such ascapped). In certain embodiments, the N-terminus of the non-naturallyoccurring cystine knot peptide (CKP) that binds to VEGF-A is modified(such as capped). In certain embodiments, both the C- and N-termini ofthe non-naturally occurring cystine knot peptide (CKP) that binds toVEGF-A are modified (such as capped). In certain embodiments, theC-terminal carboxyl group of the non-naturally occurring cystine knotpeptide (CKP) that binds to VEGF-A is amidated. In certain embodiments,the N-terminal amine of the non-naturally occurring cystine knot peptide(CKP) that binds to VEGF-A is acetylated. In certain embodiments, theC-terminal carboxyl group of the non-naturally occurring cystine knotpeptide (CKP) that binds to VEGF-A is amidated and the N-terminal amineof the non-naturally occurring cystine knot peptide (CKP) that binds toVEGF-A is acetylated.

In certain embodiments, the non-naturally occurring VEGF-A-binding CKPis a variant of a non-naturally occurring VEGF-A-binding CKP describedherein. In certain embodiments, such a variant comprises at least 1, atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, or at least 10 amino acid substitutions in one ormore of the sequences set forth in SEQ ID NOs: 8-14, 28-39, 60-69,127-129, 141, 225-230, 245-261, 287-291, 396-403, and 414; SEQ ID NOs:93-97, 117-120, 211, 298-309, and 322-339; amino acid sequences LAG,LQQ, VER, MSD, MNQ, MQT, VYQ, FIN, VSQ, VTE, FYE, MEQ, and VYR, FVR andLSN; and/or 15-18, 41-46, 71-79, 131-133, 233-238, 262-264, and 292. Incertain embodiments, the amino acid substitution(s) are conservativeamino acid substitution(s). In certain embodiments, the amino acidsubstitutions do not substantially reduce the ability of thenon-naturally occurring VEGF-A-binding CKP to bind human VEGF-A. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce VEGF-A bindingaffinity may be made. The binding affinity of a variant of anon-naturally occurring VEGF-A-binding CKP can be assessed using amethod described in the Examples below.

Conservative substitutions are shown in Table 17 below under the headingof “conservative substitutions.” More substantial changes are providedin Table 17 under the heading of “exemplary substitutions,” and asfurther described below in reference to amino acid side chain classes.Amino acid substitutions may be introduced into a variant of anon-naturally occurring VEGF-A-binding CKP and the products screened fora desired activity, e.g., retained/improved VEGF-A binding.

TABLE 17 Conservative Substitutions Original Exemplary Preferred ResidueSubstitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln;Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C)Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala AlaHis (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; LeuNorleucine Leu (L) Norleucine; Ile; Val; Met; Ala; Ile Phe Lys (K) Arg;Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala;Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W)Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe;Ala; Leu Norleucine

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

An exemplary substitutional variant is an affinity matured non-naturallyoccurring VEGF-A-binding CKP, which may be conveniently generated, e.g.,using phage display based affinity maturation techniques such as thosedescribed herein. Briefly, one or more residues in L1, L2, L3, L4,and/or L5 is altered (i.e., added, deleted, or substituted) and thevariant VEGF-A-binding CKP is displayed on phage and screened for VEGF-Abinding affinity. In certain embodiments of affinity maturation,diversity is introduced into the variable genes chosen for maturation byany of a variety of methods (e.g., error-prone PCR, loop shuffling, oroligonucleotide-directed mutagenesis). A secondary library is thencreated. The library is then screened to identify any non-naturallyoccurring CKP variants with the desired affinity for VEGF-A. In certainembodiments, introducing diversity involves loop-directed approaches, inwhich several residues in L1, L2, L3, L4, and/or L5 (e.g., about 5,about 4-6, or about 6-10 residues at a time) are randomized. L1, L2, L3,L4, and/or L5 residues involved in binding a target ligand may beidentified, e.g., using alanine scanning mutagenesis or modeling.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPYWGCGQSFECLAGCVCQYYQSCG (SEQ IDNO: 215).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCVCQYYQSCG (SEQ IDNO: 216).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPYWGCGRDFECLAKCVCQYYQSCG (SEQ IDNO: 542).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPYWGCGRDFECMSDCICQYYQSCG (SEQ IDNO: 363).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPYWGCGRDFECMSDCICQYYQSCG (SEQ IDNO: 364).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPYWGCGRDFECMNQCICQYYQSCG (SEQ IDNO: 222).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPYWGCGRDFECFYECICQYYQSCG (SEQ IDNO: 223).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPYWGCGRDFECMEQCICQYYQSCG (SEQ IDNO: 142).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNILLPFWGCGRDFECLAGCVCQYYQSCG (SEQ IDNO: 405).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNILLPYWGCGRDFECLAGCVCQYYQSCG (SEQ IDNO: 406).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMSPFWGCGRDFECLAGCVCQYYQSCG (SEQ IDNO: 407).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMTPFWGCGRDFECLAGCVCQYYQSCG (SEQ IDNO: 408).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMQPFWGCGRDFECLAGCVCQYYQSCG (SEQ IDNO: 409).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMNPFWGCGRDFECLAGCVCQYYQSCG (SEQ IDNO: 410).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMEPFWGCGRDFECLAGCVCQYYQSCG (SEQ IDNO: 411).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMDPFWGCGRDFECLAGCVCQYYQSCG (SEQ IDNO: 412).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPSWGCGRDFECLAGCVCQYYQSCG (SEQ IDNO: 415).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPFWGCGRDFECLSGCVCQYYQSCG (SEQ IDNO: 421).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPFWGCGRDFECLTGCVCQYYQSCG (SEQ IDNO: 422).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPFWGCGRDFECLEGCVCQYYQSCG (SEQ IDNO: 423).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCLCQYYQSCG (SEQ IDNO: 424).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCICQYYQSCG (SEQ IDNO: 425).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCVCQYYQSCS (SEQ IDNO: 431).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNILLPYWGCGRDFECMEQCICQYYQSCG (SEQ IDNO: 435).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCDVLQPYWGCGPDIDCLSNCICHWYNSCG (SEQ IDNO: 386).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNILLPFWGCGRDFECVSQCICQYYQSCG (SEQ IDNO: 547).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNILQPFWGCGRDFECVSQCICQYYQSCG (SEQ IDNO: 548).

In certain embodiments, one or more amino acids in the sequence of anon-naturally occurring VEGF-A binding CKP provided herein aresubstituted with unnatural amino acids. In certain embodiments, the oneor more amino acids are substituted with the same unnatural amino acid.In certain embodiments, the one or more amino acids are each substitutedwith a different unnatural amino acid. In certain embodiments, thenon-naturally occurring VEGF-A binding CKP comprises an unnatural aminoacid at any amino acid position in L1, L2, L3, L4, and/or L5, withrespect to scaffold structure I.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCVCQYYQSCG (SEQ IDNO: 216), wherein the N-terminal glycine is capped withC(═O)-oxetane-3yl.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPYWGCGRDFECLAXCVCQYYQSCG (SEQ IDNO: 568, wherein X is ornithine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence XCNIMLPYWGCGRDFECLAGCVCQYYQSCG (SEQ IDNO: 361), wherein X is N-acetylglycine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPXWGCGRDFECLAGCVCQYYQSCG (SEQ IDNO: 362), wherein X is sulfotyrosine. In certain embodiments, thenon-naturally occurring VEGF-A binding CKP comprises the amino acidsequence GCNIMLPXWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 362), wherein X is3,4-difluoro-L-phenylalanine. In certain embodiments, the non-naturallyoccurring VEGF-A binding CKP comprises the amino acid sequenceGCNIMLPXWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 362), wherein X is3,4-dichloro-L-phenylalanine. In certain embodiments, the non-naturallyoccurring VEGF-A-binding CKP comprises the amino acid sequenceGCNIMLPXWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 362), wherein X is4-chloro-L-phenylalanine. In certain embodiments, the non-naturallyoccurring VEGF-A binding CKP comprises the amino acid sequenceGCNIMLPXWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 362), wherein X is3-F,4-Cl-L-phenylalanine. In certain embodiments, the non-naturallyoccurring VEGF-A binding CKP comprises the amino acid sequenceGCNIMLPXWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 362), wherein X is 2-pyridone(NH para)-L-alanine. In certain embodiments, the non-naturally occurringVEGF-A binding CKP comprises the amino acid sequenceGCNIMLPXWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 362), wherein X is pyridone(NH meta)-L-alanine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIXLPFWGCGRDFECLAGCVCQYYQSCG (SEQ IDNO: 218), wherein X is norleucine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPFXGCGRDFECLAGCVCQYYQSCG (SEQ IDNO: 219), wherein X is 1-naphthylalanine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPFXGCGRDFECLAGCVCQYYQSCG (SEQ IDNO: 219), wherein X is 2-naphthylalanine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence XCNIMLPFWGCGRDFECLAGCVCQYYQSCG (SEQ IDNO: 221), wherein X is PEG6-propargylglycine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIXLPYWGCGRDFECMSDCICQYYQSCG (SEQ IDNO: 365), wherein X is norleucine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIXLPFWGCGRDFECMSDCICQYYQSCG (SEQ IDNO: 144), wherein X is norleucine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIXLPFWGCGRDFECVSQCICQYYQSCG (SEQ IDNO: 145), wherein X is norleucine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequenceGCNIX₁LPFWGCGRDF-D/E-CVS-N/K/X₂₋CICQYYQSCG (SEQ ID NO: 540) wherein X₁is norleucine and X₂ is ornithine. In certain embodiments, thenon-naturally occurring VEGF-A binding CKP comprises the amino acidsequence GCNIXLPFWGCGRDFECVSKCICQYYQSCG (SEQ ID NO: 545) wherein X isnorleucine. In certain embodiments, the non-naturally occurring VEGF-Abinding CKP comprises the amino acid sequenceGCNIX₁LPFWGCGRDFECVSX₂CICQYYQSCG (SEQ ID NO: 546) wherein X₁ isnorleucine and X₂ is ornithine. In certain embodiments, thenon-naturally occurring VEGF-A binding CKP comprises the amino acidsequence GCNIX₁LPFWGCGRDF-N/K/X₂-CVS-DIE-CICQYYQSCG (SEQ ID NO: 541),wherein X₁ is norleucine and X₂ is ornithine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIXLPFWGCGRDFKCVS-D/E-CICQYYQSCG(SEQ ID NO: 561, herein X is norleucine. In certain embodiments, thenon-naturally occurring VEGF-A binding CKP comprises the amino acidsequence CNIXLPFWGCGRDFKCVSDCICQYYQSCG (SEQ ID NO: 562, herein X isnorleucine. In certain embodiments, the non-naturally occurring VEGF-Abinding CKP comprises the amino acid sequenceCNIXLPFWGCGRDFKCVSECICQYYQSCG (SEQ ID NO: 563, herein X is norleucine.In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIX1LPFWGCGRDFX2CVS-DIE-CICQYYQSCG(SEQ ID NO: 564, herein X1 is norleucine and X2 is ornithine. In certainembodiments, the non-naturally occurring VEGF-A binding CKP comprisesthe amino acid sequence GCNIX1LPFWGCGRDFX2CVSDCICQYYQSCG (SEQ ID NO:565, herein X1 is norleucine and X2 is ornithine. In certainembodiments, the non-naturally occurring VEGF-A binding CKP comprisesthe amino acid sequence GCNIX1LPFWGCGRDFX2CVSECICQYYQSCG (SEQ ID NO:566, herein X1 is norleucine and X2 is ornithine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIXLPYWGCGRDFECMEQCICQYYQSCG (SEQ IDNO: 146), wherein X is norleucine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequenceGCNIX1LPYWGCGRDF-D/E-CME-N/K/X2-CICQYYQSCG (SEQ ID NO: 538) wherein X1is norleucine and X2 is ornithine. In certain embodiments, thenon-naturally occurring VEGF-A binding CKP comprises the amino acidsequence GCNIXLPYWGCGRDFECMEKCICQYYQSCG (SEQ ID NO: 543), wherein X isnorleucine. In certain embodiments, the non-naturally occurring VEGF-Abinding CKP comprises the amino acid sequenceGCNIX1LPYWGCGRDFECMEX2CICQYYQSCG (SEQ ID NO: 544), wherein X1 isnorleucine and X2 is ornithine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequenceGCNIX1LPYWGCGRDF-N/K/X2-CME-D/E-CICQYYQSCG (SEQ ID NO: 539) wherein X1is norleucine and X2 is ornithine. In certain embodiments, thenon-naturally occurring VEGF-A binding CKP comprises the amino acidsequence GCNIXLPYWGCGRDFKCME-D/E-CICQYYQSCG (SEQ ID NO: 555) wherein Xis norleucine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIXLPYWGCGRDFKCMEDCICQYYQSCG (SEQ IDNO: 556) wherein X is norleucine. In certain embodiments, thenon-naturally occurring VEGF-A binding CKP comprises the amino acidsequence GCNIXLPYWGCGRDFKCMEECICQYYQSCG (SEQ ID NO: 557) wherein X isnorleucine. In certain embodiments, the non-naturally occurring VEGF-Abinding CKP comprises the amino acid sequenceGCNIX1LPYWGCGRDFX2CME-D/E-CICQYYQSCG (SEQ ID NO: 558) wherein X1 isnorleucine and X2 is ornithine. In certain embodiments, thenon-naturally occurring VEGF-A binding CKP comprises the amino acidsequence GCNIX1LPYWGCGRDFX2CMEDCICQYYQSCG (SEQ ID NO: 559) wherein X1 isnorleucine and X2 is ornithine. In certain embodiments, thenon-naturally occurring VEGF-A binding CKP comprises the amino acidsequence GCNIX1LPYWGCGRDFX2CMEECICQYYQSCG (SEQ ID NO: 560) wherein X1 isnorleucine and X2 is ornithine.

In certain embodiments the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequenceGCDVX1QPYWGCGPDI-D/E-CLS-N/K/X2-CICHWYNSCG (SEQ ID NO: 534), wherein X1is norleucine and X2 is ornithine. In certain embodiments thenon-naturally occurring VEGF-A binding CKP comprises the amino acidsequence GCDVXQPYWGCGPDIDCLSKCICHWYNSCG (SEQ ID NO: 536), wherein X isnorleucine. In certain embodiments, the non-naturally occurring VEGF-Abinding CKP comprises the amino acid sequenceGCDVX1QPYWGCGPDIDCLSX2CICHWYNSCG (SEQ ID NO: 537), wherein X1 isnorleucine and X2 is ornithine. In certain embodiments the non-naturallyoccurring VEGF-A binding CKP comprises the amino acid sequenceGCDVX1QPYWGCGPDI-N/K/X2-CLS-D/E-CICHWYNSCG (SEQ ID NO: 535), wherein X1is norleucine and X2 is ornithine. In certain embodiments thenon-naturally occurring VEGF-A binding CKP comprises the amino acidsequence GCDVXQPYWGCGPDIDCLSNCICHWYNSCG (SEQ ID NO: 224), wherein X isnorleucine. In certain embodiments the non-naturally occurring VEGF-Abinding CKP comprises the amino acid sequenceGCDVXQPYWGCGPDIKCLS-D/E-CICHWYNSCG (SEQ ID NO: 549), wherein X isnorleucine. In certain embodiments the non-naturally occurring VEGF-Abinding CKP comprises the amino acid sequenceGCDVXQPYWGCGPDIKCLSDCICHWYNSCG (SEQ ID NO: 550), wherein X isnorleucine. In certain embodiments the non-naturally occurring VEGF-Abinding CKP comprises the amino acid sequenceGCDVXQPYWGCGPDIKCLSECICHWYNSCG (SEQ ID NO: 551), wherein X isnorleucine. In certain embodiments the non-naturally occurring VEGF-Abinding CKP comprises the amino acid sequenceGCDVX1QPYWGCGPDIX2CLS-D/E-CICHWYNSCG (SEQ ID NO: 552), wherein X1 isnorleucine and X2 is ornithine. In certain embodiments the non-naturallyoccurring VEGF-A binding CKP comprises the amino acid sequenceGCDVX1QPYWGCGPDIX2CLSDCICHWYNSCG (SEQ ID NO: 553), wherein X1 isnorleucine and X2 is ornithine In certain embodiments the non-naturallyoccurring VEGF-A binding CKP comprises the amino acid sequenceGCDVX1QPYWGCGPDIX2CLSECICHWYNSCG (SEQ ID NO: 554), wherein X1 isnorleucine and X2 is ornithine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLXFWGCGRDFECLAGCVCQYYQSCG (SEQ IDNO: 413), wherein X is gamma-benzyl-L-proline. In certain embodiments,the non-naturally occurring VEGF-A binding CKP comprises the amino acidsequence GCNIMLXFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 413), wherein X isgamma-(4-fluoro-benzyl)-L-proline. In certain embodiments, thenon-naturally occurring VEGF-A binding CKP comprises the amino acidsequence GCNIMLXFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 413), wherein X is4-OH-L-proline. In certain embodiments, the non-naturally occurringVEGF-A binding CKP comprises the amino acid sequenceGCNIMLXFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 413), wherein X is4-fluoro-L-proline. In certain embodiments, the non-naturally occurringVEGF-A binding CKP comprises the amino acid sequenceGCNIMLXFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 413), wherein X is4-[4-(trifluoromethyl)benzyl]-L-proline.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPYXGCGRDFECLAGCVCQYYQSCG (SEQ IDNO: 417), wherein X is N-methyl indole. In certain embodiments, thenon-naturally occurring VEGF-A binding CKP comprises the amino acidsequence GCNIMLPYXGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 417), wherein X isN-ethyl indole. In certain embodiments, the non-naturally occurringVEGF-A binding CKP comprises the amino acid sequenceGCNIMLPYXGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 417), wherein X isN-isopropyl indole. In certain embodiments, the non-naturally occurringVEGF-A binding CKP comprises the amino acid sequenceGCNIMLPYXGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 417), wherein X is5-aza-indole.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED IDNO: 419), wherein X is 4-methyl-L-phenylalanine. In certain embodiments,the non-naturally occurring VEGF-A binding CKP comprises the amino acidsequence GCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO: 419), wherein X is2-naphthyl-L-alanine. In certain embodiments, the non-naturallyoccurring VEGF-A binding CKP comprises the amino acid sequenceGCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO: 419), wherein X is2-quinolyl-Alanine. In certain embodiments, the non-naturally occurringVEGF-A binding CKP comprises the amino acid sequenceGCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO: 419), wherein X is4,4′-biphenyl-L-alanine. In certain embodiments, the non-naturallyoccurring VEGF-A binding CKP comprises the amino acid sequenceGCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO: 419), wherein X is3-(3-quinolinyl)-L-alanine. In certain embodiments, the non-naturallyoccurring VEGF-A binding CKP comprises the amino acid sequenceGCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO: 419), wherein X is3-(2-quinolinyl)-L-alanine. In certain embodiments, the non-naturallyoccurring VEGF-A binding CKP comprises the amino acid sequenceGCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO: 419), wherein X is3-(2-quinoxalinyl)-L-alanine. In certain embodiments, the non-naturallyoccurring VEGF-A binding CKP comprises the amino acid sequenceGCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO: 419), wherein X is4-methyl-2-pyridyl-alanine. In certain embodiments, the non-naturallyoccurring VEGF-A binding CKP comprises the amino acid sequenceGCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO: 419), wherein X is4-ethyl-2-pyridyl-L-alanine. In certain embodiments, the non-naturallyoccurring VEGF-A binding CKP comprises the amino acid sequenceGCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO: 419), wherein X isbenzothiazole-L-alanine. In certain embodiments, the non-naturallyoccurring VEGF-A binding CKP comprises the amino acid sequenceGCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO: 419), wherein X isbenzothiophene-L-alanine. In certain embodiments, the non-naturallyoccurring VEGF-A binding CKP comprises the amino acid sequenceGCNIMLPYWGCGRDXECLAGCVCQYYQSCG (SED ID NO: 419), wherein X is3-(3-isoquinolinyl)-L-alanine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPYWGCGRDFECXAGCVCQYYQSCG (SEQ IDNO: 420), wherein X is t-butyl-L-alanine (also known asL-Nepentylglycine). In certain embodiments, the non-naturally occurringVEGF-A binding CKP comprises the amino acid sequenceGCNIMLPYWGCGRDFECXAGCVCQYYQSCG (SEQ ID NO: 420), wherein X is3-cyclobutyl-L-alanine. In certain embodiments, the non-naturallyoccurring VEGF-A binding CKP comprises the amino acid sequenceGCNIMLPYWGCGRDFECXAGCVCQYYQSCG (SEQ ID NO: 420), wherein X is3-cyclopentyl-L-alanine. In certain embodiments, the non-naturallyoccurring VEGF-A binding CKP comprises the amino acid sequenceGCNIMLPYWGCGRDFECXAGCVCQYYQSCG (SEQ ID NO: 420), wherein X is5,5,5-Trifluoro-L-leucine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCXCQYYQSCG (SEQ IDNO: 426), wherein X is L-tert-Leucine. In certain embodiments, thenon-naturally occurring VEGF-A binding CKP comprises the amino acidsequence GCNIMLPYWGCGRDFECLAGCXCQYYQSCG (SEQ ID NO: 426), wherein X ist-butyl-L-alanine (also known as L-Nepentylglycine). In certainembodiments, the non-naturally occurring VEGF-A binding CKP comprisesthe amino acid sequence GCNIMLPYWGCGRDFECLAGCXCQYYQSCG (SEQ ID NO: 426),wherein X is L-cyclopentylglycine. In certain embodiments, thenon-naturally occurring VEGF-A binding CKP comprises the amino acidsequence GCNIMLPYWGCGRDFECLAGCXCQYYQSCG (SEQ ID NO: 426), wherein X is3-cyclopentyl-L-alanine. In certain embodiments, the non-naturallyoccurring VEGF-A binding CKP comprises the amino acid sequenceGCNIMLPYWGCGRDFECLAGCXCQYYQSCG (SEQ ID NO: 426), wherein X isL-cyclobutyl-L-glycine. In certain embodiments, the non-naturallyoccurring VEGF-A binding CKP comprises the amino acid sequenceGCNIMLPYWGCGRDFECLAGCXCQYYQSCG (SEQ ID NO: 426), wherein X is3-cyclobutyl-L-alanine. In certain embodiments, the non-naturallyoccurring VEGF-A binding CKP comprises the amino acid sequenceGCNIMLPYWGCGRDFECLAGCXCQYYQSCG (SEQ ID NO: 426), wherein X is5,5,5-Trifluoro-L-leucine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCVCQXYQSCG (SEQ IDNO: 428), wherein X is 2-pyridone. In certain embodiments, thenon-naturally occurring VEGF-A binding CKP comprises the amino acidsequence GCNIMLPYWGCGRDFECLAGCVCQXYQSCG (SEQ ID NO: 428), wherein X is3,4-hydroxy-L-phenylalanine. In certain embodiments, the non-naturallyoccurring VEGF-A binding CKP comprises the amino acid sequenceGCNIMLPYWGCGRDFECLAGCVCQXYQSCG (SEQ ID NO: 428), wherein X is 3,4-fluorophenylalanine. In certain embodiments, the non-naturally occurringVEGF-A binding CKP comprises the amino acid sequenceGCNIMLPYWGCGRDFECLAGCVCQXYQSCG (SEQ ID NO: 428), wherein X is3-fluoro,4-OH-L-phenylalanine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCVCQYXQSCG (SEQ IDNO: 430), wherein X is 2-Chloro-L-Tyrosine. In certain embodiments, thenon-naturally occurring VEGF-A binding CKP comprises the amino acidsequence GCNIMLPYWGCGRDFECLAGCVCQYXQSCG (SEQ ID NO: 430), wherein X is2-methyl-L-tyrosine. In certain embodiments, the non-naturally occurringVEGF-A binding CKP comprises the amino acid sequenceGCNIMLPYWGCGRDFECLAGCVCQYXQSCG (SEQ ID NO: 430), wherein X is2-ethyl-L-tyrosine, or 4-(naphthalen-1-ol-)-L-alanine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPYWGCGRDFECLAGCVCQYYQSCX (SEQ IDNO: 432), wherein X is D-serine, L-beta-homoserine, L-beta-alanine,N-alpha-methylglycine, glycine with its carboxy terminus converted to anester of glycerol, glycine with its carboxy terminus converted to anester of glycol, glycine with its carboxy terminus converted to an esterof oxetanyl alcohol, or glycine morpholine amide.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIXQPYWGCGRDFECMEQCICQYYQSCG (SEQ IDNO: 436), wherein X is norleucine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIX₁LPYWGCGRDFECX₂EQCICQYYQSCG (SEQID NO: 437). In certain embodiments, X₁ and X₂ are the same unnaturalamino acid. In certain embodiments, X₁ and X₂ are different unnaturalamino acids. In certain embodiments, X₁ and X₂ are norleucine. Incertain embodiments, X₁ is norleucine and X₂ is 3-cyclobutyl-L-alanine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIXLPYWGCGRDFECLEQCICQYYQSCG (SEQ IDNO: 438), wherein X is norleucine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIX₁LPYWGCGRDFECX₂EQCX₃CQYYQSCG (SEQID NO: 439). In certain embodiments, X₁, X₂, and/or X₃ are the sameunnatural amino acid. In certain embodiments, X₁, X₂, and/or X₃ are notthe same unnatural amino acid. In certain embodiments, X₁ is norleucine,X₂ is 3-cyclobutyl-L-alanine, and X₃ is cyclobutyl-L-glycine. In certainembodiments, X₁ is norleucine, X₂ is 3-cyclobutyl-L-alanine, and X₃ is3-cyclobutyl-L-alanine. In certain embodiments, X₁ is norleucine, X₂ is3-cyclobutyl-L-alanine, and X₃ is norleucine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence CNIX₁QPYWGCGRDFECX₂EQCX₃CQYYQSCG (SEQID NO: 440). In certain embodiments, X₁, X₂, and/or X₃ are the sameunnatural amino acid. In certain embodiments, X₁, X₂, and/or X₃ are notthe same unnatural amino acid. In certain embodiments, X₁ is norleucine,X₂ is 3-cyclobutyl-L-alanine, and X₃ is cyclobutyl-L-glycine. In certainembodiments, X₁ is norleucine, X₂ is 3-cyclobutyl-L-alanine, and X₃ is3-cyclobutyl-L-alanine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNILLPYWGCGRDFECXEQCICQYYQSCG (SEQ IDNO: 441), wherein X is 3-cyclobutyl-L-alanine or t-butyl-L-alanine (alsoknown as L-Nepentylglycine).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNILLPYWGCGRDFECMEQCXCQYYQSCG (SEQ IDNO: 442), wherein X is cyclobutyl-L-glycine or 3-cyclobutyl-L-alanine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence X₁CNIX₂LPYWGCGRDFECMEQCICQYYQSCX₃ (SEQID NO: 443).

In certain embodiments, X₁, X₂, and/or X₃ are the same unnatural aminoacid. In certain embodiments, X₁, X₂, and/or X₃ are not the sameunnatural amino acid. In certain embodiments, X₁ is N-acetylglycine, X₂is norleucine, and X₃ is glycine amide.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence X₁CNILLPYWGCGRDFECMEQCICQYYQSCX₂ (SEQID NO: 444). In certain embodiments, X₁ and X₂ are the same unnaturalamino acid. In certain embodiments, X₁ and X₂ are different unnaturalamino acids. In certain embodiments, X₁ is N-acetylglycine and X₂ isglycine amide.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence X₁CNILQPYWGCGRDFECMEQCICQYYQSCX₂ (SEQID NO: 445). In certain embodiments, X₁ and X₂ are the same unnaturalamino acid. In certain embodiments, X₁ and X₂ are different unnaturalamino acids. In certain embodiments, X₁ is N-acetylglycine and X₂ isglycine amide.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence X₁CNILQPYWGCGRDFECLEQCICQYYQSCX₂ (SEQID NO: 446). In certain embodiments, X₁ and X₂ are the same unnaturalamino acid. In certain embodiments, X₁ and X₂ are different unnaturalamino acids. In certain embodiments, X₁ is N-acetylglycine and X₂ isglycine amide.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCDVLQPYWGCGPDIDCXSNCICHWYNSCG (SEQ IDNO: 447), wherein X is 3-cyclobutyl-L-alanine or t-butyl-L-alanine(L-Nepentylglycine).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCDVLQPYWGCGPDIDCLSNCXCHWYNSCG (SEQ IDNO: 448), wherein X is cyclobutyl-L-glycine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCDVX₁QPYWGCGPDIDCX₂SNC2X₃CHWYNSCG(SEQ ID NO: 449). In certain embodiments, X₁, X₂, and/or X₃ are the sameunnatural amino acid. In certain embodiments, X₁, X₂, and/or X₃ are notthe same unnatural amino acid. In certain embodiments, X₁ is norleucine,X₂ is 3-cyclobutyl-L-alanine, and X₃ is cyclobutyl-L-glycine. In certainembodiments, X₁ is norleucine, X₂ is 3-cyclobutyl-L-alanine, and X₃ is3-cyclobutyl-L-alanine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCDVLQPYWGCGPDIDCX₁SNC2X₂CHWYNSCG (SEQID NO: 450). In certain embodiments, X₁ and X₂ are the same unnaturalamino acid. In certain embodiments, X₁ and X₂ are different unnaturalamino acids. In certain embodiments, X₁ is 3-cyclobutyl-L-alanine, andX₂ is cyclobutyl-L-glycine. In certain embodiments, X₁ is3-cyclobutyl-L-alanine, and X₂ is 3-cyclobutyl-L-alanine.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence X₁CDVLQPYWGCGPDIDCX₂SNC2X₃CHWYNSCX₄(SEQ ID NO: 451). In certain embodiments, X₁, X₂, X₃, and/or X₄ are thesame unnatural amino acid. In certain embodiments, X₁, X₂, X₃, and/or X₄are not the same unnatural amino acid. In certain embodiments, X₁ isN-acetylglycine, X₂ is 3-cyclobutyl-L-alanine, X₃ iscyclobutyl-L-glycine, and X₄ is glycine amide In certain embodiments, X₁is acetylglycine, X₂ is cyclobutyl-L-alanine, X₃ iscyclobutyl-L-alanine, and X₄ is glycine amide

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence X₁CDVX₂QPYWGCGPDIDCLSNCICHWYNSCX₃(SEQID NO: 452).

In certain embodiments, X₁, X₂, and/or X₃ are the same unnatural aminoacid. In certain embodiments, X₁, X₂, and/or X₃ are not the sameunnatural amino acid. In certain embodiments, X₁ is N-acetylglycine, X₂is norleucine, and X₃ is glycine amide

In certain embodiments, the non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence X₁CDVLQPYWGCGPDIDCLSNCICHWYNSCX₂ (SEQID NO: 453). In certain embodiments, X₁ and X₂ are the same unnaturalamino acid. In certain embodiments, X₁ and X₂ are different unnaturalamino acids. In certain embodiments, X₁ is N-acetylglycine and X₂ isglycine amide

In certain embodiments, the C-terminus of the non-naturally occurringcystine knot peptide (CKP) that binds to VEGF-A is modified (such ascapped). In certain embodiments, the N-terminus of the non-naturallyoccurring cystine knot peptide (CKP) that binds to VEGF-A is modified(such as capped). In certain embodiments, both the C- and N-termini ofthe non-naturally occurring cystine knot peptide (CKP) that binds toVEGF-A are modified (such as capped). In certain embodiments, theC-terminal carboxyl group of the non-naturally occurring cystine knotpeptide (CKP) that binds to VEGF-A is amidated. In certain embodiments,the N-terminal amine of the non-naturally occurring cystine knot peptide(CKP) that binds to VEGF-A is acetylated. In certain embodiments, theC-terminal carboxyl group of the non-naturally occurring cystine knotpeptide (CKP) that binds to VEGF-A is amidated and the N-terminal amineof the non-naturally occurring cystine knot peptide (CKP) that binds toVEGF-A is acetylated.

Structural Characteristics

In certain embodiments, the structure of a non-naturally occurringVEGF-Abinding CKP provided herein has a disulfide bond connectivity thatis different from the WT EETI-II protein, i.e., different from theC1-C4, C2-05, and C3-C6 disulfide bond pattern characteristic of WTEETI-II. In certain embodiments, a non-naturally occurring VEGF-Abinding CKP provided herein has a disulfide bond connectivity of C1-C4,C2-C3, and C5-C6. Methods of determining the disulfide bond connectivityof, e.g., a non-naturally occurring VEGF-A binding CKP, include, e.g.,by solving and analyzing the co-crystal structure of a non-naturallyoccurring VEGF-A binding CKP in complex with VEGF-A, via massspectrometry following partial reduction alkylation, or via massspectrometry following proteolytic digestion, performing structurecalculations as described in Sampoli et al. (2000) Proteins Struct FunctGen. 40, 168-174, etc.

In certain embodiments, the non-naturally occurring VEGF-A binding CKPand has an alpha helix content of at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, or at leastabout 50%, including any range in between these values. In certainembodiments, at least 7, at least 8, at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, or at least 18 amino acids of the non-naturally occurringVEGF-A binding CKP form the alpha helix. In certain embodiments, thenon-naturally occurring VEGF-A binding CKP has a disulfide bondconnectivity of C1-C4, C2-C3, and C5-C6 and an alpha helix content of atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, or at least about 50%, including any range in betweenthese values. In certain embodiments, the alpha helix content of anon-naturally occurring VEGF-A binding CKP is determined by, e.g.,circular dichroism (CD), optical rotary dispersion (ORD), nuclearmagnetic resonance (NMR), by solving and analyzing the co-crystalstructure of a non-naturally occurring VEGF-A binding CKP in complexwith VEGF-A, via mass spectrometry following partial reductionalkylation, or via mass spectrometry following proteolytic digestion.

In certain embodiments, a non-naturally occurring VEGF-A binding CKPprovided herein competes for binding to VEGF-A with a secondnon-naturally occurring VEGF-A binding CKP, wherein the secondnon-naturally occurring VEGF-A binding CKP comprises an L1 comprisingthe amino acid sequence NIMLPFWG (SEQ ID NO: 33); an L2 comprising theamino acid sequence KQDSD (SEQ ID NO: 93); an L3 comprising the aminoacid sequence LAG; an L4 comprising the amino acid V, and an L5comprising the amino acid sequence QYYQS (SEQ ID NO: 45), with referenceto scaffold structure I. In certain embodiments, the non-naturallyoccurring VEGF-A binding CKP provided herein competes for binding toVEGF-A with a second non-naturally occurring VEGF-A binding CKP, whereinthe second non-naturally occurring VEGF-A binding CKP comprises theamino acid sequence GCNIMLPFWGCKQDSDCLAGCVCQYYQSCG (SEQ ID NO: 52).

In certain embodiments, a non-naturally occurring VEGF-A binding CKPprovided herein binds the same epitope on VEGF-A bound by a secondnon-naturally occurring VEGF-A binding CKP, wherein the secondnon-naturally occurring VEGF-A binding CKP comprises an L1 comprisingthe amino acid sequence NIMLPFWG (SEQ ID NO: 33); an L2 comprising theamino acid sequence KQDSD (SEQ ID NO: 93); an L3 comprising the aminoacid sequence LAG; an L4 comprising the amino acid V, and an L5comprising the amino acid sequence QYYQS (SEQ ID NO: 45), with referenceto scaffold structure I. In certain embodiments, the non-naturallyoccurring VEGF-A binding CKP provided herein binds the same epitope onVEGF-A bound by a second non-naturally occurring VEGF-A binding CKP,wherein the second non-naturally occurring VEGF-A binding CKP comprisesthe amino acid sequence GCNIMLPFWGCKQDSDCLAGCVCQYYQSCG (SEQ ID NO: 52).

In certain embodiments, the non-naturally VEGF-A binding CKP providedherein competes for binding to VEGF-A with a second non-naturallyoccurring VEGF-A binding CKP, wherein the second VEGF-A binding CKPcomprises an L1 comprising the amino acid sequence NIMLPFWG (SEQ ID NO:33); an L2 comprising the amino acid sequence GRDFE (SEQ ID NO: 97); anL3 comprising the amino acid sequence LAG; an L4 comprising the aminoacid V, and an L5 comprising the amino acid sequence QYYQS (SEQ ID NO:45), with reference to scaffold structure I. In certain embodiments, thenon-naturally occurring VEGF-A binding CKP provided herein competes forbinding to VEGF-A with a second non-naturally occurring VEGF-A bindingCKP, wherein the second non-naturally occurring VEGF-A binding CKPcomprises the amino acid sequence GCNIMLPFWGCGRDFECLAGCVCQYYQSCG (SEQ IDNO: 102).

In certain embodiments, the non-naturally occurring VEGF-A binding CKPprovided herein binds the same epitope on VEGF-A bound by a secondnon-naturally occurring VEGF-A binding CKP, wherein the secondnon-naturally occurring VEGF-A-binding CKP comprises an L1 comprisingthe amino acid sequence NIMLPFWG (SEQ ID NO: 33); an L2 comprising theamino acid sequence GRDFE (SEQ ID NO: 97); an L3 comprising the aminoacid sequence LAG; an L4 comprising the amino acid V, and an L5comprising the amino acid sequence QYYQS (SEQ ID NO: 45), with referenceto scaffold structure I. In certain embodiments, the non-naturallyoccurring VEGF-A-binding CKP provided herein binds the same epitope onVEGF-A bound by a second non-naturally occurring VEGF-A-binding CKP,wherein the second non-naturally occurring VEGF-A-binding CKP comprisesthe amino acid sequence GCNIMLPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 102).

In certain embodiments, the non-naturally occurring VEGF-A-binding CKPprovided herein competes for binding to VEGF-A with a secondnon-naturally occurring VEGF-A-binding CKP, wherein the secondnon-naturally occurring VEGF-A-binding CKP comprises an L1 comprisingthe amino acid sequence DVMQPYWG (SEQ ID NO: 35); an L2 comprising theamino acid sequence KQDSD (SEQ ID NO: 93); an L3 comprising the aminoacid sequence LAG; an L4 comprising the amino acid V, and an L5comprising the amino acid sequence HWYNS (SEQ ID NO: 46), with referenceto scaffold structure I. In certain embodiments, the non-naturallyoccurring VEGF-A-binding CKP provided herein competes for binding toVEGF-A with a second non-naturally occurring VEGF-A-binding CKP, whereinthe second non-naturally occurring VEGF-A-binding CKP comprises theamino acid sequence GCDVMQPYWGCKQDSDCLAGCVCHWYNSCG (SEQ ID NO: 55).

In certain embodiments, the non-naturally occurring VEGF-A-binding CKPprovided herein binds the same epitope on VEGF-A bound by a secondnon-naturally occurring VEGF-A-binding CKP, wherein the secondnon-naturally occurring VEGF-A-binding CKP comprises an L1 comprisingthe amino acid sequence DVMQPYWG (SEQ ID NO: 35); an L2 comprising theamino acid sequence KQDSD (SEQ ID NO: 93); an L3 comprising the aminoacid sequence LAG; an L4 comprising the amino acid V, and an L5comprising the amino acid sequence HWYNS (SEQ ID NO: 46), with referenceto scaffold structure I. In certain embodiments, the non-naturallyoccurring VEGF-A-binding CKP provided herein binds the same epitope onVEGF-A bound by a second non-naturally occurring VEGF-A-binding CKP,wherein the second non-naturally occurring VEGF-A-binding CKP comprisesthe amino acid sequence GCDVMQPYWGCKQDSDCLAGCVCHWYNSCG (SEQ ID NO: 55).

In certain embodiments, the non-naturally occurring VEGF-A-binding CKPprotein provided herein competes for binding to VEGF-A with a secondnon-naturally occurring VEGF-A-binding CKP, wherein the secondVEGF-A-binding CKP comprises an L1 comprising the amino acid sequenceDVMQPYWG (SEQ ID NO: 35); an L2 comprising the amino acid sequence GPDID(SEQ ID NO: 118); an L3 comprising the amino acid sequence LAG; an L4comprising the amino acid V, and an L5 comprising the amino acidsequence HWYNS (SEQ ID NO: 46), with reference to scaffold structure I.In certain embodiments, the non-naturally occurring VEGF-A-binding CKPprovided herein competes for binding to VEGF-A with a secondnon-naturally occurring VEGF-A-binding CKP, wherein the secondnon-naturally occurring VEGF-A-binding CKP comprises the amino acidsequence GCDVMQPYWGCGPDIDCLAGCVCHWYNSCG (SEQ ID NO: 123).

In certain embodiments, the non-naturally occurring VEGF-A-binding CKPprovided herein binds the same epitope on VEGF-A bound by a secondnon-naturally occurring VEGF-A-binding CKP, wherein the secondnon-naturally occurring VEGF-A-binding CKP comprises an L1 comprisingthe amino acid sequence DVMQPYWG (SEQ ID NO: 35); an L2 comprising theamino acid sequence GPDID (SEQ ID NO: 118); an L3 comprising the aminoacid sequence LAG; an L4 comprising the amino acid V, and an L5comprising the amino acid sequence HWYNS (SEQ ID NO: 46), with referenceto scaffold structure I. In certain embodiments, the non-naturallyoccurring VEGF-A-binding CKP provided herein binds the same epitope onVEGF-A bound by a second non-naturally occurring VEGF-A-binding CKP,wherein the second non-naturally occurring VEGF-A-binding CKP comprisesthe amino acid sequence GCDVMQPYWGCGPDIDCLAGCVCHWYNSCG (SEQ ID NO: 123).

In certain embodiments, a non-naturally occurring VEGF-A-binding CKPprovided herein binds an epitope of VEGF-A comprising at least one, atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, at least nine, at least ten, or morethan ten amino acids selected from the group consisting of V14, V15,F17, D19, Y21, Q22, Y25, I46, K48, N62, D63, L66, M81, 183, K84, P85,H86, Q87, G88, Q89, I91, C104, R105, and P106. In certain embodiments, anon-naturally occurring VEGF-A-binding CKP provided herein binds anepitope of VEGF-A comprising K48, N62, and D63. In certain embodiments,a non-naturally occurring VEGF-A-binding CKP provided herein binds anepitope of VEGF-A comprising H86. In certain embodiments, non-naturallyoccurring VEGF-A-binding CKP provided herein binds an epitope of VEGF-Acomprising Y21, Y25, and P106. In certain embodiments, a non-naturallyoccurring VEGF-A-binding CKP provided herein binds an epitope of VEGF-Acomprising M81, D19, and Q22. In certain embodiments, a non-naturallyoccurring VEGF-A-binding CKP provided herein binds an epitope of VEGF-Acomprising F17, M81, and I91. In certain embodiments, non-naturallyoccurring VEGF-A-binding CKP provided herein binds an epitope of VEGF-Acomprising V14, F17, D19, Q22, M81, and I91. In certain embodiments, anon-naturally occurring VEGF-A-binding CKP provided herein binds anepitope of VEGF-A comprising Q22 and Y25.

In certain embodiments, a non-naturally occurring VEGF-A-binding CKPprovided herein binds an epitope on VEGF-A that overlaps the epitope ofVEGF-A bound by the anti-VEGF-A antibody G6.31 (Fuh et al. (2006) J.Biol. Chem. 281, 6625-6631). In certain embodiments, a non-naturallyoccurring VEGF-A-binding CKP provided herein binds an epitope on VEGF-Athat overlaps with the epitope of VEGF-A bound by Flt-1. In certainembodiments, a non-naturally occurring VEGF-A-binding CKP providedherein binds an epitope on VEGF-A that overlaps with the epitope ofVEGF-A bound by bevacizumab.

Functional Characteristics

In certain embodiments, a non-naturally occurring CKP that “specificallybinds” VEGF-A (such as a human VEGF-A, a mouse VEGF-A, and/or a ratVEGF-A) has a binding affinity (Kd) value of no more than about 1×10⁻⁷M, preferably no more than about 1×10⁻⁸ and most preferably no more thanabout 1×10⁻⁹ M) but has a binding affinity for a homologue of VEGF-A orother growth factor which is at least about 50-fold, or at least about500-fold, or at least about 1000-fold, weaker than its binding affinityfor VEGF-A.

In certain embodiments, the extent of binding of a non-naturallyoccurring VEGF-A-binding CKP provided herein to, e.g., a non-targetprotein (e.g., a homolog of VEGFA such as VEGF-B, VEGF-C and VEGF-D) orother growth factors (such as P1GF, EGF, NGF, IGF and PDGF) is less thanabout 10% of the binding of the non-naturally occurring VEGF-A-bindingCKP to VEGF-A as determined by methods known in the art, such as ELISA,fluorescence activated cell sorting (FACS) analysis, orradioimmunoprecipitation (RIA). Specific binding can be measured, forexample, by determining binding of a molecule compared to binding of acontrol molecule, which generally is a molecule of similar structurethat does not have binding activity. For example, specific binding canbe determined by competition with a control molecule that is similar tothe target, for example, an excess of non-labeled target. In this case,specific binding is indicated if the binding of the labeled target to aprobe is competitively inhibited by excess unlabeled target. Othermethods of assessing the binding of a non-naturally occurring CKP that“specifically binds” VEGF-A are described in the Examples.

The term “specific binding” or “specifically binds to” or is “specificfor” a particular polypeptide or an epitope on a particular polypeptidetarget as used herein can be exhibited, for example, by a moleculehaving a Kd for the target of at least about 10⁻⁴ M, alternatively atleast about 10⁻⁵ M, alternatively at least about 10⁻⁶ M, alternativelyat least about 10⁻⁷ M, alternatively at least about 10⁻⁸ M,alternatively at least about 10⁻⁹ M, alternatively at least about 10⁻¹⁰M, alternatively at least about 10⁻¹¹ M, alternatively at least about10⁻¹² M, or greater. In one embodiment, the term “specific binding”refers to binding where a molecule binds to a particular polypeptide orepitope on a particular polypeptide without substantially binding to anyother polypeptide or polypeptide epitope.

In certain embodiments, the non-naturally occurring VEGF-A-binding CKPbinds VEGF-A with a Kd between about 1 pM to about 500 nM. In certainembodiments, the non-naturally occurring VEGF-A-binding CKP binds VEGF-Awith a Kd between about 1 pM to about 50 pM, between about 50 pM toabout 250 pM, between about 250 pM to about 500 pM, between about 500 pMto 750 pM, between about 750 pM to about 1 nM, between about 1 nM toabout 25 nM, between about 25 nM to about 50 nM, between 50 nM to about100 nM, between about 100 nM to about 250 nM, or between about 250 nM toabout 500 nM.

In certain embodiments, the non-naturally occurring VEGF-A-binding CKPbinds human VEGF-A, a mouse VEGF-A, and/or a rat VEGF-A. In certainembodiments, non-naturally occurring VEGF-A-binding CKP that binds humanVEGF-A, a mouse VEGF-A, and a rat VEGF-A comprises an L1 comprising theamino acid sequence NIMLPFWG (SEQ ID NO: 33); an L2 comprising the aminoacid sequence KQDSD (SEQ ID NO: 93); an L3 comprising the amino acidsequence LAG; an L4 comprising the amino acid V, and an L5 comprisingthe amino acid sequence QYYQS (SEQ ID NO: 45), with reference toscaffold structure I. In certain embodiments, the non-naturallyoccurring VEGF-A-binding CKP that binds human VEGF-A, a mouse VEGF-A,and a rat VEGF-A comprises the amino acid sequenceGCNIMLPFWGCKQDSDCLAGCVCQYYQSCG (SEQ ID NO: 52).

In certain embodiments, non-naturally occurring VEGF-A-binding CKP bindshuman VEGF-A, a mouse VEGF-A, and a rat VEGF-A comprises an L1comprising the amino acid sequence NIMLPFWG (SEQ ID NO: 33); an L2comprising the amino acid sequence GRDFE (SEQ ID NO: 97); an L3comprising the amino acid sequence LAG; an L4 comprising the amino acidV, and an L5 comprising the amino acid sequence QYYQS (SEQ ID NO: 45),with reference to scaffold structure I. In certain embodiments, thenon-naturally occurring VEGF-A-binding CKP that binds human VEGF-A, amouse VEGF-A, and a rat VEGF-A comprises the amino acid sequenceGCNIMLPFWGCGRDFECLAGCVCQYYQSCG (SEQ ID NO: 102).

In certain embodiments, non-naturally occurring VEGF-A-binding CKP thatbinds human VEGF-A, a mouse VEGF-A, and a rat VEGF-A comprises an L1comprising the amino acid sequence DVMQPYWG (SEQ ID NO: 35); an L2comprising the amino acid sequence KQDSD (SEQ ID NO: 93); an L3comprising the amino acid sequence LAG; an L4 comprising the amino acidV, and an L5 comprising the amino acid sequence HWYNS (SEQ ID NO: 46),with reference to scaffold structure I. In certain embodiments, thenon-naturally occurring VEGF-A-binding CKP that binds human VEGF-A, amouse VEGF-A, and a rat VEGF-A comprises the amino acid sequenceGCDVMQPYWGCKQDSDCLAGCVCHWYNSCG (SEQ ID NO: 55).

In certain embodiments, non-naturally occurring VEGF-A-binding CKP thatbinds human VEGF-A, a mouse VEGF-A, and a rat VEGF-A comprises an L1comprising the amino acid sequence DVMQPYWG (SEQ ID NO: 35); an L2comprising the amino acid sequence GPDID (SEQ ID NO: 118); an L3comprising the amino acid sequence LAG; an L4 comprising the amino acidV, and an L5 comprising the amino acid sequence HWYNS (SEQ ID NO: 46),with reference to scaffold structure I. In certain embodiments, thenon-naturally occurring VEGF-A-binding CKP that binds human VEGF-A, amouse VEGF-A, and a rat VEGF-A comprises the amino acid sequenceGCDVMQPYWGCGPDIDCLAGCVCHWYNSCG (SEQ ID NO: 123).

In certain embodiments, the non-naturally occurring VEGF-A-binding CKPdescribed herein has an IC₅₀ value of less than about 0.5 nM, less thanabout 0.6 nM, less than about 0.7 nM, less than about 0.8 nM, less thanabout 0.9 nM, or less than about 1.0 nM, including any range in betweenthese values.

In certain embodiments, the non-naturally occurring VEGF-A-binding CKPdoes not inhibit trypsin protease activity as measured in a peptidesubstrate cleavage assay (e.g., the peptide substrate cleavage assaydescribed in the Examples).

In certain embodiments, the non-naturally occurring VEGF-A-binding CKPis resistant to trypsin digestion. In certain embodiments, about 30% orless, about 25% or less, or about 20% or less of the non-naturallyoccurring VEGF-A-binding CKP is cleaved at Arg13 within loop 2 after 24h incubation with trypsin at 37° C.

Nucleic acid molecules encoding non-naturally occurring VEGF-A-bindingCKPs described herein, expression vectors comprising nucleic acidmolecules encoding the non-naturally occurring VEGF-A-binding CKP, andcells comprising the nucleic acid molecules are also contemplated. Alsoprovided herein are methods of producing a non-naturally occurringVEGF-A-binding CKP described herein by culturing such cells, expressingthe non-naturally occurring VEGF-A-binding CKP, and recovering thenon-naturally occurring VEGF-A-binding CKP from the cell culture.

In certain embodiments, a non-naturally occurring VEGF-A-binding CKP isproduced via in vitro translation, as described elsewhere herein.

In certain embodiments, a non-naturally occurring VEGF-A-binding CKP isgenerated via chemical peptide synthesis, e.g., by grafting chemicallysynthesized L1, L2, L3, L4, and/or L5 peptides onto an scaffoldframework (such as scaffold structure I), or by chemically synthesizingthe entire non-naturally occurring VEGF-A-binding CKP.

Non-Naturally Occurring Cystine Knot Peptides (CKPs) that Bind Human LowDensity Lipoprotein Receptor (LDL)-Related Protein 6 (LRP6)

LDL receptors are transmembrane cell surface proteins involved inreceptor-mediated endocytosis of lipoprotein and protein ligands. HumanLDL receptor-related protein 6 (LRP6) (Accession Nos: NM_002336 (mRNA)and NP_002327 (protein); UniProtKB: O75581) functions as a receptor or,with Frizzled, a co-receptor for Wnt and thereby transmits the canonicalWnt/beta-catenin signaling cascade (Katoh et al. (2007) Clin Cancer Res13:4042-4045). Through its interaction with the Wnt/beta-cateninsignaling cascade, LRP6 plays a role in the regulation of celldifferentiation, proliferation, and migration, and in the development ofmany cancer types (Li et al. (2004) Oncogene 23:9129-9135; Tung et al.(2012) PLoS ONE 7(5): e36565. doi:10.1371/journal.pone.0036565; Liu etal. (2010) Proc Natl Acad Sci USA 107:5136-5141).

Wnt signaling is involved in many biological pathways. With respect todiseases it is involved with cancer and metastatic disease, osteoporosisand other bone metabolism and disease, neuronal and neurodegenerativedisease, rheumatoid arthritis and other inflammatory disease. Thisinhibition of Wnt signaling by blockade of LRP6 may have a wide range oftherapeutic utility. Bone loss is a serious medical problem, not onlyduring postmenopausal osteoporosis, but also in rheumatoid arthritis.Bone is degraded in multiple myeloma and in bone metastases. Therapeuticstrategies aimed at strengthening bone, fracture prevention, orrestoration of damaged bone are therefore of very high interest (Kawaiet al. (2011) Nat. Rev. Drug Discov. 10, 141-156; Mason and Williams(2010) J. Osteoporosis, vol. 2010, Article ID 460120, 9 pages;doi:10.4061/2010/460120). The Wnt pathway inhibitors DKK1 and SOST,because of their roles in suppressing new bone formation, are consideredhighly promising therapeutic targets; antibodies with neutralizing thefunction of SOST show significant preclinical activity (Ominsky et al.(2010) J. Bone Miner. Res. 25, 948-959) and are now in human clinicaltrials (Padhi et al. (2011) J. Bone Miner. Res. 26, 19-26.

Misregulated Wnt signaling is implicated in diseases ranging fromosteoporosis to cancer (Clevers (2006) Cell 127: 469-80; MacDonald etal. 2009. Dev Cell 17: 9-26; Nusse (2008) Cell Res 18: 523-7; Polakis(2007) Curr Opin Genet Dev 17: 45-51). This list has expanded to includemetabolic disorders (Mani et al. (2007) Science 315: 1278-82 andneurodegeneration (Caricasole et al. (2004) J Neurosci 24: 6021-7; DeFerrari et al. (2007) Proc Natl Acad Sci USA 104: 9434-9). An especiallyclear link exists between mutations of the protein adenomatous polyposiscoli (APC), which prevent effective regulation of β-catenin levels, andcolorectal cancers (Polakis (2007) Curr Opin Genet Dev 17: 45-51). Alsoof particular note is the strong genetic relationship between LRP5 andbone homeostasis. Loss-of-function mutations in LRP5 cause the autosomalrecessive disorder osteoporosispseudoglioma syndrome (OPPG),characterized by low bone mass, ocular defects and a predisposition tofractures (Gong et al. (2001) Cell 107: 513-23.

Provided herein is a non-naturally occurring CKP that binds to human lowdensity lipoprotein receptor-related protein 6 (LRP6), wherein thenon-naturally CKP comprises the following cystine scaffold structure(i.e., scaffold structure I):

Z₁C₁L1C₂L2C₃L3C₄L4C₅L5C₆Z₂  (I)

wherein:

Z₁ and Z₂ are any amino acid;

L1 is Loop 1 and has a structure selected from the group consisting of:X₁X₂X₃X₄X₅X₆, X₁X₂X₃X₄X₅X₆X₇, X₁X₂X₃X₄X₅X₆X₇X₈, X₁X₂X₃X₄X₅X₆X₇X₈X₉, andX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀, wherein each of X₁-X₁₀ is any amino acid;

L2 is Loop 2 and has the structure: X₁X₂X₃X₄X₅, wherein each of X₁-X₅ isany amino acid;

L3 is Loop 3 and has the structure: X₁X₂X₃ wherein each of X₁-X₃ is anyamino acid;

L4 is Loop 4 and has the structure: X₁, wherein X₁ is any amino acid;and

L5 is Loop 5 and has the structure: X₁X₂X₃X₄X₅, wherein each of X₁-X₅ isany amino acid.

In certain embodiments, Z₁ and/or Z₂ of the non-naturally occurringcystine knot peptide (CKP) that binds to LRP6 is G. In certainembodiments, Z₁ and/or Z₂ comprise more than one amino acid. In certainembodiments, Z₁ and/or Z₂ comprise 4 amino acids. In certainembodiments, Z₁ and/or Z₂ comprise 5 amino acids.

In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an L1 wherein X₁ is an amino acid selected from R, V, M, A, G,N, S, and E; wherein X₂ is an amino acid selected from T, N, S, G, R,and A; wherein X₃ is an amino acid selected from N, R, H, V, K, S, G, I,and Y; wherein X₄ is an amino acid selected from R, V, N, I, K, S, andT; wherein X₅ is an amino acid selected from V, R, K, I, T, S, L, and N;and wherein X₆ is an amino acid selected from K, G, A, I, R, N, S, andV. In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an L1 wherein X₇ is an amino acid selected from G, R, K, E, P,and T. In certain embodiments, the non-naturally occurring LRP6-bindingCKP comprises an L1 wherein X₈ is an amino acid selected from G, R, K,Q, A, and S. In certain embodiments, the non-naturally occurringLRP6-binding CKP comprises an L1 wherein X₉ is an amino acid selectedfrom R or G. In certain embodiments, the non-naturally occurringLRP6-binding CKP comprises an L1 wherein X₁₀ is an amino acid selectedfrom E, W, and G. In certain embodiments, the non-naturally occurringLRP6-binding CKP comprises an L5 wherein X₁ is an amino acid selectedfrom G, S, N, Y, A, and R; wherein X₂ is an amino acid selected from P,G, S, V, E, R, F, and D; wherein X₃ is an amino acid selected from N, G,S, E, P, K, H, and R; wherein X₄ is an amino acid selected from G, R, H,S, Q, V, and D; and wherein X₅ is an amino acid selected from F, D, N,R, G, Y, S, and T. In certain embodiments, the non-naturally occurringLRP6-binding CKP comprises an L2 wherein X₁ is K, X₂ is Q, X₃ is D, X₄is S, and X₅ is D. In certain embodiments, the non-naturally occurringLRP6-binding CKP comprises an L3 wherein X₁ is L, X₂ is A, and X₃ is G.In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an L4 wherein X₁ is V.

In certain embodiments, the non-naturally occurring LRP6-binding CKPcompetitively inhibits the binding of a competing molecule to humanLRP6. In certain embodiments, the competing molecule is an anti-LRP6antibody. In certain embodiments, the competing molecule is a secondnon-naturally occurring LRP6-binding CKP.

Non-naturally occurring LRP6-binding CKPs that bind to overlapping orsimilar areas on a target can be identified by competitiveinhibition/binding assays. Such assays are well known in the art and aredescribed in, e.g., S. J. Mather (ed.) 1996. Current Directions inRadiopharmaceutical Research and Development, 169-179, Kluwer AcademicPublishers; Zettner (1973) Clin. Chem. 19, 699-705; Gao (2012)Analytical Methods 4, 3718-3723.

In certain embodiments, the non-naturally occurring LRP6-binding CKPbinds the same epitope of human LRP6 bound by a second non-naturallyoccurring LRP6-binding CKP comprising an L1 that comprises the aminoacid sequenceV/R/N/S/E/G-N/S/G/R-R/V/K/S/N/I/Y-V/N/I/R/S/T-R/K/I/N-G/I/R/K/S/A (SEQID NO: 185) orA/R/M/V/G/S-N/T/S/A-R/N/H-V/R/K-K/V/I-R/K/A/N/S/V-T/G/R/K/P-S/G/R/A (SEQID NO: 186) orR/A/Q-S/A-G/S/N/I-N/K-T/S/L/R-I/R/V-R/E/K-K/Q/A/R-R/G/Q-E/W/G/R (SEQ IDNO: 187); an L2 that comprises the amino acid sequence KQDSD (SEQ ID NO:93); an L3 that comprises the amino acid sequence LAG; an L4 thatcomprises the amino acid V; and an L5 that comprises the amino acidsequenceG/S/N/Y/A/R-P/G/S/V/E/R/F/D-N/G/S/E/P/K/H/R-G/R/H/S/Q/V/D-F/D/N/R/G/Y/S/T(SEQ ID NO: 188), with reference to scaffold structure I.

In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an L1 that comprises the amino acid sequenceV/R/N/S/E/G-N/S/G/R-R/V/K/S/N/I/Y-V/N/I/R/S/T-R/K/I/N-G/I/R/K/S/A (SEQID NO: 185) orA/R/M/V/G/S-N/T/S/A-R/N/H-V/R/K-K/V/I-R/K/A/N/S/V-T/G/R/K/P-S/G/R/A (SEQID NO: 186) or R/A-S-G/S/N-N/K-T/S/L-I/R-R/E-K/Q/A-R/G-E/W/G (SEQ ID NO:187)), with reference to scaffold structure I. In certain embodiments,the non-naturally occurring LRP6-binding CKP further comprises an L2that comprises the amino acid sequence KQDSD (SEQ ID NO: 93). In certainembodiments, the non-naturally occurring LRP6-binding CKP furthercomprises an L3 that comprises the amino acid sequence LAG. In certainembodiments, the non-naturally occurring LRP6-binding CKP furthercomprises an L4 that comprises the amino acid V. In certain embodiments,the non-naturally occurring LRP6-binding CKP further comprises an L5that comprises the amino acid sequenceG/S/N/Y/A/R-P/G/S/V/E/R/F/D-N/G/S/E/P/K/H/R-G/R/H/S/Q/V/D-F/D/N/R/G/Y/S/T(SEQ ID NO: 188

In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an L1 and/or L5 of any one of the non-naturally occurringLRP6-binding CKPs disclosed herein. In certain embodiments, thenon-naturally occurring LRP6-binding CKP comprises an L1 that comprisesan amino acid sequence set forth in any one of SEQ ID NOs: 147-168 and367, with respect to scaffold structure I. In certain embodiments, thenon-naturally occurring LRP6-binding CKP further comprises an L2 thatcomprises the amino acid sequence set forth in SEQ ID NO: 93. In certainembodiments, the non-naturally occurring LRP6-binding CKP furthercomprises an L3 that comprises the amino acid sequence LAG. In certainembodiments, the non-naturally occurring LRP6-binding CKP furthercomprises an L4 comprising the amino acid V. In certain embodiments, thenon-naturally occurring LRP6-binding CKP further comprises an L5 thatcomprises an amino acid sequence set forth in any one of SEQ ID NOs: 19and 169-184.

The L1 and L5 amino acid sequences described above are provided in Table18 below:

TABLE 18 RTNRVKGG (SEQ ID NO: 147) GPNGF (SEQ ID NO: 19)VNRVRG (SEQ ID NO: 148) SGGRD (SEQ ID NO: 169) MNHVKARR (SEQ ID NO: 149)GPNGF (SEQ ID NO: 19) RSVNKI (SEQ ID NO: 150) GSSRN (SEQ ID NO: 170)VNKIKG (SEQ ID NO: 151) GVEGR (SEQ ID NO: 171) RNSIKR (SEQ ID NO: 152)SVGHG (SEQ ID NO: 172) VSNRVNKG (SEQ ID NO: 153) GPNGF (SEQ ID NO: 19)RGNIIK (SEQ ID NO: 154) NESRG (SEQ ID NO: 173)RSGNTIRKRE (SEQ ID NO: 155) GGPGG (SEQ ID NO: 174)ASSNSIRQGW (SEQ ID NO: 156) GPKSN (SEQ ID NO: 175)RSNRIR (SEQ ID NO: 157) YGHGD (SEQ ID NO: 176)RSNKLREARG (SEQ ID NO: 158) GSRQD (SEQ ID NO: 177)VNSVKR (SEQ ID NO: 159) SRGVN (SEQ ID NO: 178) GSNKIRPR (SEQ ID NO: 160)GPNDF (SEQ ID NO: 179) NRIRNS (SEQ ID NO: 161) GRGDY (SEQ ID NO: 180)SRNSIK (SEQ ID NO: 162) ASGSS (SEQ ID NO: 181) SNYVKR (SEQ ID NO: 163)SPGGR (SEQ ID NO: 182) RANRVSGR (SEQ ID NO: 164) GPNGF (SEQ ID NO: 19)SNRVKVRA (SEQ ID NO: 165) GPNGF (SEQ ID NO: 19) ENRTKG (SEQ ID NO: 166)GFRGT (SEQ ID NO: 183) GNKIRA (SEQ ID NO: 167) RDRVG (SEQ ID NO: 184)ANRVKRTS (SEQ ID NO: 168) GPNGF (SEQ ID NO: 19)QAINRVKRQR (SEQ ID NO: 367) V/R/N/S/E/G-N/S/G/R- A/R/M/M/G/S-N/T/S/A-R/V/K/S/N/I/Y-V/N/I/R/S/T- R/N/H-V/R/K-K/V/I- R/K/I/N-G/I/R/K/S/AR/K/A/N/S/V-T/G/R/K/ (SEQ ID NO: 185) P-S/G/R/A  (SEQ ID NO: 186)R/A-S-G/S/N-N/K-T/S/L-I/R- G/S/N/Y/A/R-P/G/S/V/E/ R/E-K/Q/A-R/G-E/W/G R/F/D-N/G/S/E/P/K/H/R- (SEQ ID NO: 187) G/R/H/S/Q/V/D-F/D/N/R/ G/Y/S/T(SEQ ID NO: 188)

In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an L1 that comprises the amino acid sequence RTNRVKGG (SEQ IDNO: 147); an L2 that comprises the amino acid sequence KQDSD (SEQ ID NO:93); an L3 that comprises the amino acid sequence LAG; an L4 thatcomprises the amino acid V; and an L5 that comprises the amino acidsequence GPNGF (SEQ ID NO: 19), with reference to with reference toscaffold structure I.

In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an L1 that comprises the amino acid sequence VNRVRG (SEQ IDNO: 148); an L2 that comprises the amino acid sequence KQDSD (SEQ ID NO:93); an L3 that comprises the amino acid sequence LAG; an L4 thatcomprises the amino acid V; and an L5 that comprises the amino acidsequence SGGRD (SEQ ID NO: 169), with reference to with reference toscaffold structure I.

In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an L1 that comprises the amino acid sequence MNHVKARR (SEQ IDNO: 149); an L2 that comprises the amino acid sequence KQDSD (SEQ ID NO:93); an L3 that comprises the amino acid sequence LAG; an L4 thatcomprises the amino acid V; and an L5 that comprises the amino acidsequence GPNGF (SEQ ID NO: 19), with reference to scaffold structure I.

In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an L1 that comprises the amino acid sequence RSVNKI (SEQ IDNO: 150); an L2 that comprises the amino acid sequence KQDSD (SEQ ID NO:93); an L3 that comprises the amino acid sequence LAG; an L4 thatcomprises the amino acid V; and an L5 that comprises the amino acidsequence GSSRN (SEQ ID NO: 170), with reference to scaffold structure I.

In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an L1 that comprises the amino acid sequence VNKIKG (SEQ IDNO: 151); an L2 that comprises the amino acid sequence KQDSD (SEQ ID NO:93); an L3 that comprises the amino acid sequence LAG; an L4 thatcomprises the amino acid V; and an L5 that comprises the amino acidsequence GVEGR (SEQ ID NO: 29), with reference to scaffold structure I.

In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an L1 that comprises the amino acid sequence RNSIKR (SEQ IDNO: 152); an L2 that comprises the amino acid sequence KQDSD (SEQ ID NO:93); an L3 that comprises the amino acid sequence LAG; an L4 thatcomprises the amino acid V; and an L5 that comprises the amino acidsequence SVGHG (SEQ ID NO: 172), with reference to scaffold structure I.

In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an L1 that comprises the amino acid sequence VSNRVNKG (SEQ IDNO: 153); an L2 that comprises the amino acid sequence KQDSD (SEQ ID NO:93); an L3 that comprises the amino acid sequence LAG; an L4 thatcomprises the amino acid V; and an L5 that comprises the amino acidsequence GPNGF (SEQ ID NO: 19), with reference to scaffold structure I.

In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an L1 that comprises the amino acid sequence RGNIIK (SEQ IDNO: 154); an L2 that comprises the amino acid sequence KQDSD (SEQ ID NO:93); an L3 that comprises the amino acid sequence LAG; an L4 thatcomprises the amino acid V; and an L5 that comprises the amino acidsequence NESRG (SEQ ID NO: 173), with reference to scaffold structure I.

In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an L1 that comprises the amino acid sequence RSGNTIRKRE (SEQID NO: 155); an L2 that comprises the amino acid sequence KQDSD (SEQ IDNO: 93); an L3 that comprises the amino acid sequence LAG; an L4 thatcomprises the amino acid V; and an L5 that comprises the amino acidsequence GGPGG (SEQ ID NO: 174), with reference to scaffold structure I.

In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an L1 that comprises the amino acid sequence ASSNSIRQGW (SEQID NO: 156); an L2 that comprises the amino acid sequence KQDSD (SEQ IDNO: 93); an L3 that comprises the amino acid sequence LAG; an L4 thatcomprises the amino acid V; and an L5 that comprises the amino acidsequence GPKSN (SEQ ID NO: 175), with reference to scaffold structure I.

In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an L1 that comprises the amino acid sequence RSNRIR (SEQ IDNO: 157); an L2 that comprises the amino acid sequence KQDSD (SEQ ID NO:93); an L3 that comprises the amino acid sequence LAG; an L4 thatcomprises the amino acid V; and an L5 that comprises the amino acidsequence YGHGD (SEQ ID NO: 176), with reference to scaffold structure I.

In certain embodiments, non-naturally occurring LRP6-binding CKP an L1that comprises the amino acid sequence RSNKLREARG (SEQ ID NO: 158); anL2 that comprises the amino acid sequence KQDSD (SEQ ID NO: 93); an L3that comprises the amino acid sequence LAG; an L4 that comprises theamino acid V; and an L5 that comprises the amino acid sequence GSRQD(SEQ ID NO: 177), with reference to scaffold structure I.

In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an L1 that comprises the amino acid sequence VNSVKR (SEQ IDNO: 159); an L2 that comprises the amino acid sequence KQDSD (SEQ ID NO:93); an L3 that comprises the amino acid sequence LAG; an L4 thatcomprises the amino acid V; and an L5 that comprises the amino acidsequence SRGVN (SEQ ID NO: 178), with reference to scaffold structure I.

In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an L1 that comprises the amino acid sequence GSNKIRPR (SEQ IDNO: 160); an L2 that comprises the amino acid sequence KQDSD (SEQ ID NO:93); an L3 that comprises the amino acid sequence LAG; an L4 thatcomprises the amino acid V; and an L5 that comprises the amino acidsequence GPNDF (SEQ ID NO: 179), with reference to scaffold structure I.

In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an L1 that comprises the amino acid sequence NRIRNS (SEQ IDNO: 161); an L2 that comprises the amino acid sequence KQDSD (SEQ ID NO:93); an L3 that comprises the amino acid sequence LAG; an L4 thatcomprises the amino acid V; and an L5 that comprises the amino acidsequence GRGDY (SEQ ID NO: 180), with reference to scaffold structure I.

In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an L1 that comprises the amino acid sequence SRNSIK (SEQ IDNO: 162); an L2 that comprises the amino acid sequence KQDSD (SEQ ID NO:93); an L3 that comprises the amino acid sequence LAG; an L4 thatcomprises the amino acid V; and an L5 that comprises the amino acidsequence ASGSS (SEQ ID NO: 181), with reference to scaffold structure I.

In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an L1 that comprises the amino acid sequence SNYVKR (SEQ IDNO: 163); an L2 that comprises the amino acid sequence KQDSD (SEQ ID NO:93); an L3 that comprises the amino acid sequence LAG; an L4 thatcomprises the amino acid V; and an L5 that comprises the amino acidsequence SPGGR (SEQ ID NO: 182), with reference to scaffold structure I.

In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an L1 that comprises the amino acid sequence RANRVSGR (SEQ IDNO: 164); an L2 that comprises the amino acid sequence KQDSD (SEQ ID NO:93); an L3 that comprises the amino acid sequence LAG; an L4 thatcomprises the amino acid V; and an L5 that comprises the amino acidsequence GPNGF (SEQ ID NO: 19), with reference to scaffold structure I.

In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an L1 that comprises the amino acid sequence SNRVKVRA (SEQ IDNO: 165); an L2 that comprises the amino acid sequence KQDSD (SEQ ID NO:93); an L3 that comprises the amino acid sequence LAG; an L4 thatcomprises the amino acid V; and an L5 that comprises the amino acidsequence GPNGF (SEQ ID NO: 19), with reference to scaffold structure I.

In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an L1 that comprises the amino acid sequence ENRTKG (SEQ IDNO: 166); an L2 that comprises the amino acid sequence KQDSD (SEQ ID NO:93); an L3 that comprises the amino acid sequence LAG; an L4 thatcomprises the amino acid V; and an L5 that comprises the amino acidsequence GFRGT (SEQ ID NO: 183), with reference to with reference toscaffold structure I.

In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an L1 that comprises the amino acid sequence GNKIRA (SEQ IDNO: 167); an L2 that comprises the amino acid sequence KQDSD (SEQ ID NO:93); an L3 that comprises the amino acid sequence LAG; an L4 thatcomprises the amino acid V; and an L5 that comprises the amino acidsequence RDRVG (SEQ ID NO: 184), with reference to scaffold structure I.

In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an L1 that comprises the amino acid sequence ANRVKRTS (SEQ IDNO: 168); an L2 that comprises the amino acid sequence KQDSD (SEQ ID NO:93); an L3 that comprises the amino acid sequence LAG; an L4 thatcomprises the amino acid V; and an L5 that comprises the amino acidsequence GPNGF (SEQ ID NO: 19), with reference to scaffold structure I.

In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an L1 that comprises the amino acid sequence QAINRVKRQR (SEQID NO: 367); an L2 that comprises the amino acid sequence KQDSD (SEQ IDNO: 93); an L3 that comprises the amino acid sequence LAG; an L4 thatcomprises the amino acid V; and an L5 that comprises the amino acidsequence GPNGF (SEQ ID NO: 19), with reference to scaffold structure I.

In certain embodiments, the non-naturally occurring LRP6-binding CKPcomprises an amino acid sequence set forth in any one of SEQ ID NOs:189-210 and 366. SEQ ID NOs: 189-210 and 366 are provided below.

GCRTNRVKGGCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 189)GCVNRVRGCKQDSDCLAGCVCSGGRDCG (SEQ ID NO: 190)GCMNHVKARRCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 191)GCRSVNKICKQDSDCLAGCVCGSSRNCG (SEQ ID NO: 192)GCVNKIKGCKQDSDCLAGCVCGVEGRCG (SEQ ID NO: 193)GCRNSIKRCKQNSDCLAGCVCSVGHGCG (SEQ ID NO: 194)GCVSNRVNKGCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 195)GCRGNIIKCKQDSDCLAGCVCNESRGCG (SEQ ID NO: 196)GCRSGNTIRKRECKQDSDCLAGCVCGGPGGCG (SEQ ID NO: 197)GCASSNSIRQGWCKQDSDCLAGCVCGPKSNCG (SEQ ID NO: 198)GCRSNRIRCKQDSDCLAGCVCYGHGDCG (SEQ ID NO: 199)GCRSNKLREARGCKQDSDCLAGCVCGSRQDCG (SEQ ID NO: 200)GCVNSVKRCKQDSDCLAGCVCSRGVNCG (SEQ ID NO: 201)GCGSNKIRPRCKQDSDCLAGCVCGPNDFCG (SEQ ID NO: 202)GCNRIRNSCKQDSDCLAGCVCGRGDYCG (SEQ ID NO: 203)GCSRNSIKCKQDSDCLAGCVCASGSSCG (SEQ ID NO: 204)GCSNYVKRCKQDSDCLAGCVCSPGGRCG (SEQ ID NO: 205)GCRANRVSGRCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 206)GCSNRVKVRACKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 207)GCENRTKGCKQDSDCLAGCVCGFRGTCG (SEQ ID NO: 208)GCGNKIRACKQDSDCLAGCVCRDRVGCG (SEQ ID NO: 209)GCANRVKRTSCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 210)GCQAINRVKRQRCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 366)

In certain embodiments, the non-naturally occurring LRP6-binding CKP isa variant of a non-naturally occurring LRP6-binding CKP describedherein. In certain embodiments, such a variant comprises at least 1, atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, or at least 10 amino acid substitutions in one ormore of the sequences set forth in SEQ ID NOs: 19, 93, 147-168, 169-184,and 189-210 and/or in the amino acid sequence LAG. In certainembodiments, the amino acid substitution(s) are conservative amino acidsubstitution(s). In certain embodiments, the amino acid substitutions donot substantially reduce the ability of the non-naturally occurringLRP6-binding CKP to bind human LRP6. For example, conservativealterations (e.g., conservative substitutions as provided herein) thatdo not substantially reduce LRP6 binding affinity may be made. Thebinding affinity of a variant of a non-naturally occurring LRP6-bindingCKP can be assessed using a method described in the Examples below.

Conservative substitutions are shown in Table 17 above under the headingof “conservative substitutions.” More substantial changes are providedin Table 17 under the heading of “exemplary substitutions,” and asfurther described below in reference to amino acid side chain classes.Amino acid substitutions may be introduced into a variant of anon-naturally occurring LRP6-binding CKP and the products screened for adesired activity, e.g., retained/improved LRP6 binding.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. An exemplary substitutional variant isan affinity matured non-naturally occurring LRP6-binding CKP, which maybe conveniently generated, e.g., using phage display based affinitymaturation techniques such as those described herein. Briefly, one ormore residues in L1, L2, L3, L4, and/or L5 is altered (i.e., added,deleted, or substituted) and the variant LRP6-binding CKP is displayedon phage and screened for LRP6 binding affinity. In certain embodimentsof affinity maturation, diversity is introduced into the variable geneschosen for maturation by any of a variety of methods (e.g., error-pronePCR, loop shuffling, or oligonucleotide-directed mutagenesis). Asecondary library is then created. The library is then screened toidentify any non-naturally occurring CKP variants with the desiredaffinity for LRP6. In certain embodiments, introducing diversityinvolves loop-directed approaches, in which several residues in L1, L2,L3, L4, and/or L5 (e.g., about 5, about 4-6, or about 6-10 residues at atime) are randomized. L1, L2, L3, L4, and/or L5 residues involved inbinding a target ligand may be identified, e.g., using alanine scanningmutagenesis or modeling.

In certain embodiments, a non-naturally occurring CKP that “specificallybinds” human LRP6 (i.e., has a binding affinity (Kd) value of no morethan about 1×10⁻⁷ M, preferably no more than about 1×10⁻⁸ and mostpreferably no more than about 1×10⁻⁹ M) but has a binding affinity foranother LRP protein which is at least about 50-fold, or at least about500-fold, or at least about 1000-fold, weaker than its binding affinityfor LRP6.

In certain embodiments, the extent of binding of the non-naturallyoccurring LRP6-binding CKP to a non-target protein (e.g., a LRP6 homologsuch as LRP1, LRP1B, LRP2, LRP3, LRP4, LRP5, LRP8, LRP10, LRP11, andLRP12) is less than about 10% of the binding of the non-naturallyoccurring LRP6-binding CKP to human LRP6 as determined by methods knownin the art, such as ELISA, fluorescence activated cell sorting (FACS)analysis, or radioimmunoprecipitation (RIA). Specific binding can bemeasured, for example, by determining binding of a molecule compared tobinding of a control molecule, which generally is a molecule of similarstructure that does not have binding activity. For example, specificbinding can be determined by competition with a control molecule that issimilar to the target, for example, an excess of non-labeled target. Inthis case, specific binding is indicated if the binding of the labeledtarget to a probe is competitively inhibited by excess unlabeled target.The term “specific binding” or “specifically binds to” or is “specificfor” a particular polypeptide or an epitope on a particular polypeptidetarget as used herein can be exhibited, for example, by a moleculehaving a Kd for the target of at least about 10⁻⁴ M, alternatively atleast about 10⁻⁵ M, alternatively at least about 10⁻⁶ M, alternativelyat least about 10⁻⁷ M, alternatively at least about 10⁻⁸ M,alternatively at least about 10⁻⁹ M, alternatively at least about 10⁻¹⁰M, alternatively at least about 10⁻¹¹ M, alternatively at least about10⁻¹² M, or greater. In one embodiment, the term “specific binding”refers to binding where a molecule binds to a particular polypeptide orepitope on a particular polypeptide without substantially binding to anyother polypeptide or polypeptide epitope.

In certain embodiments, the non-naturally occurring LRP6-binding CKPbinds a human LRP6 with a Kd between about 1 pM to about 500 nM. Incertain embodiments, the non-naturally occurring LRP6-binding CKPprotein that specifically binds LRP6 binds a human LRP6 with a Kdbetween about 1 pM to about 50 pM, between about 50 pM to about 250 pM,between about 250 pM to about 500 pM, between about 500 pM to 750 pM,between about 750 pM to about 1 nM, between about 1 nM to about 25 nM,between about 25 nM to about 50 nM, between 50 nM to about 100 nM,between about 100 nM to about 250 nM, or between about 250 nM to about500 nM, including any range in between these values.

In certain embodiments, the non-naturally occurring LRP6-binding CKPinhibits Wnt1 signaling, e.g., as determined using methods described inthe Examples below.

Nucleic acid molecules encoding the non-naturally occurring LRP6-bindingCKPs described, expression vectors comprising nucleic acid moleculesencoding the non-naturally occurring LRP6-binding CKPs, and cellscomprising the nucleic acid molecules are also contemplated. Alsoprovided herein are methods of producing a non-naturally occurringLRP6-binding CKP by culturing such cells, expressing the non-naturallyoccurring LRP6-binding CKP, and recovering the non-naturally occurringLRP6-binding CKP from the cell culture.

In certain embodiments, a non-naturally occurring LRP6-binding CKP isproduced via in vitro translation, as described elsewhere herein.

As described elsewhere herein, a non-naturally occurring LRP6-bindingCKP is generated via chemical peptide synthesis, e.g., by graftingchemically synthesized L1, L2, L3, L4, and/or L5 peptides onto anEETI-II framework, or by chemically synthesizing the entirenon-naturally occurring LRP6-binding CKP.

In certain embodiments, the non-naturally occurring LRP6-binding CKP isas a therapeutic agent in the treatment of diseases or conditionswherein excessive LRP6 activity is involved.

Methods of Production

In certain embodiments, a non-naturally occurring VEGF-A-binding CKP ora non-naturally occurring LRP6-binding CKP is generated via geneticengineering. A variety of methods for mutagenesis have been previouslydescribed (along with appropriate methods for screening or selection).Such mutagenesis methods include, but are not limited to, e.g.,error-prone PCR, loop shuffling, or oligonucleotide-directedmutagenesis, random nucleotide insertion or other methods prior torecombination. Further details regarding these methods are described in,e.g., Abou-Nadler et al. (2010) Bioengineered Bugs 1, 337-340; Firth etal. (2005) Bioinformatics 21, 3314-3315; Cirino et al. (2003) MethodsMol Biol 231, 3-9; Pirakitikulr (2010) Protein Sci 19, 2336-2346;Steffens et al. (2007) J. Biomol Tech 18, 147-149; and others.Accordingly, in certain embodiments, provided is a non-naturallyoccurring VEGF-A-binding CKP or a non-naturally occurring LRP6-bindingCKP generated via genetic engineering techniques.

In certain embodiments, a non-naturally occurring VEGF-A-binding CKP ora non-naturally occurring LRP6-binding CKP is generated via in vitrotranslation. Briefly, in vitro translation entails cloning theprotein-coding sequence(s) into a vector containing a promoter,producing mRNA by transcribing the cloned sequence(s) with an RNApolymerase, and synthesizing the protein by translation of this mRNA invitro, e.g., using a cell-free extract. A desired variant protein can begenerated simply by altering the cloned protein-coding sequence. ManymRNAs can be translated efficiently in wheat germ extracts or in rabbitreticulocyte lysates. Further details regarding in vitro translation aredescribed in, e.g., Hope et al. (1985) Cell 43, 177-188; Hope et al.(1986) Cell 46, 885-894; Hope et al. (1987) EMBO J. 6, 2781-2784; Hopeet al. (1988) Nature 333, 635-640; and Melton et al. (1984) Nucl. AcidsRes. 12, 7057-7070.

Accordingly, provided are nucleic acid molecules encoding anon-naturally occurring VEGF-A-binding CKP or a non-naturally occurringLRP6-binding CKP described herein. An expression vector operably linkedto a nucleic acid molecule encoding a non-naturally occurringVEGF-A-binding CKP or a non-naturally occurring LRP6-binding CKP is alsoprovided. Host cells (including, e.g., prokaryotic host cells such as E.coli, eukaryotic host cells such as yeast cells, mammalian cells, CHOcells, etc.) comprising a nucleic acid encoding a non-naturallyoccurring VEGF-A-binding CKP or a non-naturally occurring LRP6-bindingCKP are also provided.

In certain embodiments, non-naturally occurring VEGF-A-binding CKP or anon-naturally occurring LRP6-binding CKP is generated via in vitrotranslation. Briefly, in vitro translation entails cloning theprotein-coding sequence(s) into a vector containing a promoter,producing mRNA by transcribing the cloned sequence(s) with an RNApolymerase, and synthesizing the protein by translation of this mRNA invitro, e.g., using a cell-free extract. A desired mutant protein can begenerated simply by altering the cloned protein-coding sequence. ManymRNAs can be translated efficiently in wheat germ extracts or in rabbitreticulocyte lysates. Further details regarding in vitro translation aredescribed in, e.g., Hope et al. (1985) Cell 43, 177-188; Hope et al.(1986) Cell 46, 885-894; Hope et al. (1987) EMBO J. 6, 2781-2784; Hopeet al. (1988) Nature 333, 635-640; and Melton et al. (1984) Nucl. AcidsRes. 12, 7057-7070.

In certain embodiments, a non-naturally occurring VEGF-A-binding CKP ora non-naturally occurring LRP6-binding CKP is generated via chemicalsynthesis. In certain embodiments, chemically synthesized L1, L2, L3,L4, and/or L5 peptides are grafted onto an EETI-II-based framework (suchas scaffold structure I) to generate non-naturally occurringVEGF-A-binding CKP or a non-naturally occurring LRP6-binding CKP. Incertain embodiments the entire non-naturally occurring VEGF-A-bindingCKP or the entire non-naturally occurring LRP6-binding CKP is chemicallysynthesized. Methods of solid phase and liquid phase peptide synthesisare well known in the art and described in detail in, e.g., Methods ofMolecular Biology, 35, Peptide Synthesis Protocols, (M. W. Penningtonand B. M. Dunn Eds), Springer, 1994; Welsch et al. (2010) Curr Opin ChemBiol 14, 1-15; Methods of Enzymology, 289, Solid Phase PeptideSynthesis, (G. B. Fields Ed.), Academic Press, 1997; Chemical Approachesto the Synthesis of Peptides and Proteins, (P. Lloyd-Williams, F.Albericio, and E. Giralt Eds), CRC Press, 1997; Fmoc Solid Phase PeptideSynthesis, A Practical Approach, (W. C. Chan, P. D. White Eds), OxfordUniversity Press, 2000; Solid Phase Synthesis, A Practical Guide, (S. F.Kates, F Albericio Eds), Marcel Dekker, 2000; P. Seneci, Solid-PhaseSynthesis and Combinatorial Technologies, John Wiley & Sons, 2000;Synthesis of Peptides and Peptidomimetics (M. Goodman, Editor-in-chief,A. Felix, L. Moroder, C. Tmiolo Eds), Thieme, 2002; N. L. Benoiton,Chemistry of Peptide Synthesis, CRC Press, 2005; Methods in MolecularBiology, 298, Peptide Synthesis and Applications, (J. Howl Ed) HumanaPress, 2005; and Amino Acids, Peptides and Proteins in OrganicChemistry, Volume 3, Building Blocks, Catalysts and Coupling Chemistry,(A. B. Hughs, Ed.) Wiley-VCH, 2011.

Chimeric Molecules Comprising a Non-Naturally Occurring EETI-II Protein

A non-naturally occurring CKP described herein (such as a non-naturallyoccurring VEGF-A-binding CKP or a non-naturally occurring LRP6-bindingCKP) can also be modified if advantageous in a way to form a chimericmolecule comprising the non-naturally occurring CKP fused (e.g.,recombinantly fused) to another, heterologous polypeptide or amino acidsequence. In certain embodiments, such a chimeric molecule comprises afusion of a non-naturally occurring CKP described herein (such as anon-naturally occurring VEGF-A-binding CKP or a non-naturally occurringLRP6-binding CKP) with an antibody to form, e.g., a divalent molecule ora bispecific molecule.

In certain embodiments, a chimeric molecule comprises a fusion of anon-naturally occurring CKP described herein (such as a non-naturallyoccurring VEGF-A-binding CKP or a non-naturally occurring LRP6-bindingCKP) with a second moiety (such as a protein transduction domain) whichtargets the chimeric molecule for delivery to various tissues, or, e.g.,across brain blood barrier, using, for example, the protein transductiondomain of human immunodeficiency virus TAT protein (Schwarze et al.,1999, Science 285: 1569-72).

In certain embodiments, the non-naturally occurring CKP provided hereincan be used as bi- or multi-specific (for different target ligands ordifferent epitopes on the same target ligand) in multimer form. Forexample, a dimeric bispecific non-naturally occurring CKP has onesubunit with specificity for a first target protein or epitope and asecond subunit with specificity for a second target protein or epitope.Non-naturally occurring CKP protein subunits can be joined in a varietyof conformations that can increase the valency and thus the avidity ofbinding to a target ligand.

In certain embodiments a chimeric molecule provided herein comprises twoor more (such as three, four, five, six, seven, eight, nine, ten, ormore than ten) non-naturally occurring CKP proteins. In certainembodiments, a nucleic acid can be engineered to encode two or morecopies of a single non-naturally occurring CKP, which copies aretranscribed and translated in tandem to produce a covalently linkedmultimer of identical subunits. In certain embodiments, the nucleic acidcan be engineered to encode two or more different non-naturallyoccurring CKPs, which copies are transcribed and translated in tandem toproduce a covalently linked multimer of different subunits that bind,e.g., different epitopes of a single target ligand, or, e.g., differenttarget ligands.

In another embodiment, such a chimeric molecule comprises a fusion of anon-naturally occurring CKP described herein (such as a non-naturallyoccurring VEGF-A-binding CKP or a non-naturally occurring LRP6-bindingCKP) with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the non-naturally occurringCKP. The presence of such epitope-tagged forms of the non-naturallyoccurring CKP protein can be detected using an antibody against the tagpolypeptide. Also, provision of the epitope tag enables thenon-naturally occurring CKP to be readily purified by affinitypurification using an anti-tag antibody or another type of affinitymatrix that binds to the epitope tag. Various tag polypeptides and theirrespective antibodies are known in the art. Examples includepoly-histidine (poly-His) or poly-histidine-glycine (poly-His-Gly) tags;the flu HA tag polypeptide and its antibody 12CA5 (Field et al. (1988)Mol. Cell. Biol. 8, 2159-2165); the c-myc tag and the 8F9, 3C7, 6E10,G4, B7 and 9E10 antibodies thereto (Evan et al. (1985) Mol. Cell. Biol.5, 3610-3616]; and the Herpes Simplex virus glycoprotein D (gD) tag andits antibody (Paborsky et al. (1990) Protein Eng., 3, 547-553). Othertag polypeptides include the Flag-peptide (Hopp et al. (1988)BioTechnology, 6, 1204-1210); the KT3 epitope peptide (Martin et al.(1992) Science, 255, 192-194]; an α-tubulin epitope peptide (Skinner etal. (1991) J. Biol. Chem. 266, 15163-15166); and the T7 gene 10 proteinpeptide tag (Lutz-Freyermuth et al. (1990) Proc. Natl. Acad. Sci. USA87, 6393-6397].

In certain embodiments, the chimeric molecule can comprise a fusion of anon-naturally occurring CKP protein described herein (such as anon-naturally occurring VEGF-A-binding CKP or a non-naturally occurringLRP6-binding CKP) with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule (e.g., an“immunoadhesin”), such a fusion could be to the Fc region of an IgGmolecule. Ig fusions provided herein include polypeptides that compriseapproximately or only residues 94-243, residues 33-53 or residues 33-52of human in place of at least one variable region within an Ig molecule.In a particularly preferred embodiment, the immunoglobulin fusionincludes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regionsof an IgG1 molecule. For the production of immunoglobulin fusions seealso, U.S. Pat. No. 5,428,130 issued Jun. 27, 1995. In certainembodiments, a non-naturally occurring CKP described herein (such as anon-naturally occurring VEGF-A-binding CKP or a non-naturally occurringLRP6-binding CKP) is fused, e.g., at the N or C terminus, to theconstant region of an IgG (Fc). In certain embodiments, thenon-naturally occurring CKP/Fc fusion molecule activates the complementcomponent of the immune response. In certain embodiments, thenon-naturally occurring CKP/Fc fusion protein increases the therapeuticvalue of the non-naturally occurring CKP. In certain embodiments, anon-naturally occurring CKP protein described herein (such as anon-naturally occurring VEGF-A-binding CKP or a non-naturally occurringLRP6-binding CKP) is fused (such as recombinantly fused), e.g., at the Nor C terminus, to a complement protein, such as C1q. Variouspublications describe methods for obtaining non-naturally occurringproteins whose half-lives are modified either by introducing anFcRn-binding polypeptide into the molecules (WO 1997/43316, U.S. Pat.No. 5,869,046, U.S. Pat. No. 5,747,035, WO 1996/32478, WO 1991/14438) orby fusing the proteins with antibodies whose FcRn-binding affinities arepreserved but affinities for other Fc receptors have been greatlyreduced (WO 1999/43713) or fusing with FcRn binding domains ofantibodies (WO 2000/09560, U.S. Pat. No. 4,703,039). Specific techniquesand methods of increasing half-life of physiologically active molecules(e.g., non-naturally occurring CKP) can also be found in U.S. Pat. No.7,083,784. In certain embodiments, a non-naturally occurring CKP proteindescribed herein (such as a non-naturally occurring VEGF-A-binding CKPor a non-naturally occurring LRP6-binding CKP) is fused to an Fc regionfrom an IgG that comprises amino acid residue mutations (as numbered bythe EU index in Kabat): M252Y/S254T/T256E or H433K/N434F/Y436H.

In certain embodiments, non-naturally occurring CKP proteins describedherein (such as a non-naturally occurring VEGF-A-binding CKP or anon-naturally occurring LRP6-binding CKP) are fused with molecules thatincrease or extend in vivo or serum half-life. In certain embodiments, anon-naturally occurring CKP described herein (such as a non-naturallyoccurring VEGF-A-binding CKP or a non-naturally occurring LRP6-bindingCKP) is fused with albumin, such as human serum albumin (HSA),polyethylene glycol (PEG), polysaccharides, immunoglobulin molecules(IgG), complement, hemoglobin, a binding peptide, lipoproteins or otherfactors to increase its half-life in the bloodstream and/or its tissuepenetration.

Additional chimeric molecules comprising non-naturally occurringVEGF-A-binding CKPs or non-naturally occurring LRP6-binding CKPs may begenerated through the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”). DNA shuffling may be employed to alter the activities ofthe non-naturally occurring CKPs (e.g., non-naturally occurring CKPswith higher affinities and lower dissociation rates). See, generally,U.S. Pat. No. 5,605,793, U.S. Pat. No. 5,811,238, U.S. Pat. No.5,830,721, U.S. Pat. No. 5,834,252, U.S. Pat. No. 5,837,458, Patten etal. (1997) Curr. Opinion Biotechnol. 8, 724-33; Harayama (1998) TrendsBiotechnol. 16, 76-82; Hansson, et al., (1999) J. Mol. Biol. 287,265-76; and Lorenzo and Blasco, (1998) Biotechniques 24, 308-313

In certain embodiments, a non-naturally occurring VEGF-A-binding CKP ora non-naturally occurring LRP6-binding CKP provided herein is altered bybeing subjected to random mutagenesis by error-prone PCR, randomnucleotide insertion or other methods prior to recombination. One ormore portions of a polynucleotide encoding a scaffold that binds to aspecific target may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules.

Any of these fusions can generated by standard techniques, for example,by expression of the fusion protein from a recombinant fusion geneconstructed using publicly available gene sequences, or by chemicalpeptide synthesis.

Conjugates Comprising a Non-Naturally Occurring VEGF-A-Binding CKP or aNon-Naturally Occurring LRP6-Binding CKP)

Provided herein are immunoconjugates comprising a non-naturallyoccurring CKP described herein (such as a non-naturally occurringVEGF-A-binding CKP or a non-naturally occurring LRP6-binding CKP)conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), Momordica charantia inhibitor, curcin, crotin, Saponariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. Other toxins include maytansine andmaytansinoids, calicheamicin and other cytotoxic agents. A variety ofradionuclides are available for the production of radioconjugatednon-naturally occurring CKPs. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re.

Conjugates of a non-naturally occurring CKP described herein (such as anon-naturally occurring VEGF-A-binding CKP or a non-naturally occurringLRP6-binding CKP) and, e.g., cytotoxic agent, are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bisdiazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionuclide to a non-naturally occurring CKP providedherein. See, WO94/11026.

In another embodiment, the non-naturally occurring CKP described herein(such as a non-naturally occurring VEGF-A-binding CKP or a non-naturallyoccurring LRP6-binding CKP) can be conjugated to a “receptor” (such asstreptavidin) for utilization in ocular “pre-targeting” wherein thenon-naturally occurring EETI-II scaffold protein-receptor conjugate isadministered to the eye patient, followed by removal of unboundconjugate from the circulation using a clearing agent and thenadministration of a “ligand” (e.g., avidin) that is conjugated to acytotoxic agent (e.g., a radionuclide) or a therapeutic agent.

In certain embodiments, the non-naturally occurring CKPs provided herein(such as a non-naturally occurring VEGF-A-binding CKP or a non-naturallyoccurring LRP6-binding CKP) can be used as bi- or multi-specific (fordifferent target ligands or different epitopes on the same targetligand) in multimer form. The attachments may be covalent ornon-covalent. For example, a dimeric bispecific non-naturally occurringCKP has one subunit with specificity for a first target protein orepitope and a second subunit with specificity for a second targetprotein or epitope. Non-naturally occurring CKP subunits can be joined,e.g., via conjugation, in a variety of conformations that can increasethe valency and thus the avidity of binding to a target ligand or tobind multiple target ligands.

In certain embodiments, non-naturally occurring CKPs provided herein areengineered to provide reactive groups for conjugation. In certainembodiments, the N-terminus and/or C-terminus may also serve to providereactive groups for conjugation. In certain embodiments, the N-terminusis conjugated to one moiety (such as, but not limited to PEG) while theC-terminus is conjugated to another moiety (such as, but not limited tobiotin), or vice versa.

Provided is a non-naturally occurring CKP described herein (such as anon-naturally occurring VEGF-A-binding CKP or a non-naturally occurringLRP6-binding CKP) conjugated to one or more moieties, including but notlimited to, peptides, polypeptides, proteins, fusion proteins, nucleicacid molecules, small molecules, mimetic agents, synthetic drugs,inorganic molecules, and organic molecules. Also provided is the use ofa non-naturally occurring CKP described herein (such as a non-naturallyoccurring VEGF-A-binding CKP or a non-naturally occurring LRP6-bindingCKP) chemically conjugated (including both covalent and non-covalentconjugations) to a heterologous protein or polypeptide (or fragmentthereof, to a polypeptide of at least 10, at least 20, at least 30, atleast 40, at least 50, at least 60, at least 70, at least 80, at least90 or at least 100 amino acids). The fusion does not necessarily need tobe direct, but may occur through linker sequences described herein.

In certain embodiments, a non-naturally occurring CKP described herein(such as a non-naturally occurring VEGF-A-binding CKP or a non-naturallyoccurring LRP6-binding CKP), or analogs or derivatives thereof may beconjugated to a diagnostic or detectable agent. Such non-naturallyoccurring CKP conjugates can be useful for monitoring or prognosing thedevelopment or progression of a disease as part of a clinical testingprocedure, such as determining the efficacy of a particular therapy.Such diagnosis and detection can be accomplished by coupling thenon-naturally occurring CKP to detectable substances including, but notlimited to various enzymes, such as but not limited to horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; prosthetic groups, such as but not limited tostreptavidinlbiotin and avidin/biotin; fluorescent materials, such asbut not limited to, umbelliferone, fluorescein, fluoresceinisothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; luminescent materials, such as, but notlimited to, luminol; bioluminescent materials, such as but not limitedto, luciferase, luciferin, and aequorin; radioactive materials, such asbut not limited to iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C), sulfur(³⁵S), tritium (³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In,), andtechnetium (⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium(¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu,¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr,¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se,¹¹³Sn, and ¹¹⁷Tn; positron emitting metals using various positronemission tomographies, nonradioactive paramagnetic metal ions, andmolecules that are radiolabeled or conjugated to specific radioisotopes.

Also provided is a non-naturally occurring CKPs (such as a non-naturallyoccurring VEGF-A-binding CKP or a non-naturally occurring LRP6-bindingCKP) conjugated to a therapeutic moiety. In certain embodiments, anon-naturally occurring CKP may be conjugated to a therapeutic moietysuch as a cytotoxin, e.g., a cytostatic or cytocidal agent, atherapeutic agent or a radioactive metal ion, e.g., alpha-emitters. Acytotoxin or cytotoxic agent includes any agent that is detrimental tocells.

In certain embodiments, a non-naturally occurring CKP described herein(such as a non-naturally occurring VEGF-A-binding CKP or a non-naturallyoccurring LRP6-binding CKP is conjugated to therapeutic moieties such asa radioactive metal ion, such as alpha-emitters such as ²¹³Bi ormacrocyclic chelators useful for conjugating radiometal ions, includingbut not limited to, ¹³¹In, ¹³¹Lu, ¹³¹Y, ¹³¹Ho, ¹³¹Sm, to polypeptides.In certain embodiments, the macrocyclic chelator is 1, 4, 7,10-tetraazacyclododecane-N,N′,N″,N′″-tetra-acetic acid (DOTA) which canbe attached to the non-naturally occurring CKP via a linker molecule.Such linker molecules are commonly known in the art and described in,e.g., Denardo et al. (1998) Clin Cancer Res. 4, 2483-90; Peterson et al.(1999) Bioconjug. Chem. 10, 553-557; and Zimmerman et al. (1999) Nucl.Med. Biol. 26, 943-50.

Techniques for conjugating therapeutic moieties to antibodies are wellknown and can be applied to the non-naturally CKPs disclosed herein,see, e.g., Amon et al., “Monoclonal Antibodies For Immunotargeting OfDrugs In Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy,Reisfeld et al. (eds.), pp. 243-56. (Alan R. Liss, Inc. 1985); Hellstromet al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2ndEd.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987);Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: AReview”, in Monoclonal Antibodies 84: Biological And ClinicalApplications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis,Results, And Future Prospective Of The Therapeutic Use Of Radio labeledAntibody In Cancer Therapy”, in Monoclonal Antibodies For CancerDetection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press1985), and Thorpe et al., 1982, Immunol. Rev. 62:119-58. Similarapproaches may be adapted for use with the non-naturally occurring CKPsprovided herein.

The therapeutic moiety or drug conjugated to a non-naturally CKPdescribed herein (such as a non-naturally occurring VEGF-A-binding CKPor a non-naturally occurring LRP6-binding CKP) should be chosen toachieve the desired prophylactic or therapeutic effect(s) for aparticular disorder in a subject. A clinician or other medical personnelshould consider the following when deciding on which therapeutic moietyor drug to conjugate to a scaffold: the nature of the disease, theseverity of the disease, and the condition of the subject.

In certain embodiments, non-naturally occurring CKPs described herein(such as a non-naturally occurring VEGF-A-binding CKP or a non-naturallyoccurring LRP6-binding CKP) can also be attached to solid supports,which are particularly useful for immunoassays or purification of thetarget antigen. Such solid supports include, but are not limited to,glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chlorideor polypropylene.

Covalent Modifications

Covalent modifications of non-naturally occurring CKPs described herein(such as a non-naturally occurring VEGF-A-binding CKP or a non-naturallyoccurring LRP6-binding CKP) are also contemplated. One type of covalentmodification includes reacting targeted amino acid residues of anon-naturally occurring CKP with an organic derivatizing agent that iscapable of reacting with selected side chains or the N- or C-terminalresidues of the non-naturally occurring CKP. Derivatization withbifunctional agents is useful, for instance, for crosslinking thenon-naturally occurring CKP to a water-insoluble support matrix orsurface for use in the method for purifying a target ligand, andvice-versa. Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidyl-propionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)-dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains (T.E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of a non-naturally occurring CKPcomprises linking the non-naturally occurring CKP to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol, or polyoxyalkylenes, in the manner set forth inU.S. Pat. No. 4,640,835, U.S. Pat. No. 4,496,689, U.S. Pat. No.4,301,144, U.S. Pat. No. 4,670,417, U.S. Pat. No. 4,791,192 or U.S. Pat.No. 4,179,337

The term “polyethylene glycol” or “PEG” means a polyethylene glycolcompound or a derivative thereof, with or without coupling agents,coupling or activating moieties (e.g., with thiol, triflate, tresylate,azirdine, oxirane, N-hydroxysuccinimide or a maleimide moiety). The term“PEG” is intended to indicate polyethylene glycol of a molecular weightbetween 500 and 150,000 Da, including analogues thereof, wherein forinstance the terminal OR-group has been replaced by a methoxy group(referred to as mPEG).

In certain embodiments, non-naturally occurring CKPs described herein(such as a non-naturally occurring VEGF-A-binding CKP or a non-naturallyoccurring LRP6-binding CKP) are derivatized with polyethylene glycol(PEG). PEG is a linear, water-soluble polymer of ethylene oxiderepeating units with two terminal hydroxyl groups. PEGs are classifiedby their molecular weights which typically range from about 500 daltonsto about 40,000 daltons. In a presently preferred embodiment, the PEGsemployed have molecular weights ranging from 5,000 daltons to about20,000 daltons. PEGs coupled to the non-naturally occurring CKPsdescribed herein can be either branched or unbranched (for example,Monfardini, C. et al. 1995 Bioconjugate Chem 6:62-69). PEGs arecommercially available from Nektar Inc., Sigma Chemical Co. and othercompanies. Such PEGs include, but are not limited to,monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethyleneglycol-succinate (MePEG-S), monomethoxypolyethylene glycol-succinimidylsuccinate (MePEG-S-NHS), monomethoxypolyethylene glycol-amine(MePEG-NH2), monomethoxypolyethylene glycol-tresylate (MePEG-TRES), andmonomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM).

In certain embodiments, the hydrophilic polymer which is employed, forexample, PEG, is capped at one end by an unreactive group such as amethoxy or ethoxy group. Thereafter, the polymer is activated at theother end by reaction with a suitable activating agent, such as cyanurichalides (for example, cyanuric chloride, bromide or fluoride),diimadozle, an anhydride reagent (for example, a dihalosuccinicanhydride, such as dibromosuccinic anhydride), acyl azide,p-diazoiumbenzyl ether, 3-(p-diazoniumphenoxy)-2-hydroxypropylether) andthe like. The activated polymer is then reacted with a non-naturallyoccurring CKP herein (such as a non-naturally occurring VEGF-A-bindingCKP or a non-naturally occurring LRP6-binding CKP) to produce anon-naturally occurring CKP derivatized with a polymer. Alternatively, afunctional group in the non-naturally occurring CKP provided herein canbe activated for reaction with the polymer, or the two groups can bejoined in a concerted coupling reaction using known coupling methods. Itwill be readily appreciated that the non-naturally occurring CKPsprovided herein can be derivatized with PEG using a myriad of otherreaction schemes known to and used by those of skill in the art.

Liposomes

Non-naturally occurring CKPs disclosed herein (such as a non-naturallyoccurring VEGF-A-binding CKP or a non-naturally occurring LRP6-bindingCKP) can also be formulated as liposomes. Liposomes containing anon-naturally occurring EETI-II scaffold protein described herein can beprepared by methods known in the art, such as described in Epstein etal., Proc Natl Acad Sci USA, 82: 3688 (1985); Hwang et al., Proc NatlAcad Sci USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and4,544,545. Liposomes with enhanced circulation time are disclosed inU.S. Pat. No. 5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. A second therapeutic agent is optionally also contained withinthe liposome. See, Gabizon et al., J. National Cancer Inst., 81(19):1484 (1989). Pharmaceutical Compositions and Formulations ComprisingNon-Naturally Cystine Knot Peptides (CKPs) That Bind Human VascularEndothelial Growth Factor A(VEGF-A)

In certain embodiments, provided herein is a pharmaceutical compositioncomprising a non-naturally occurring VEGF-A-binding CKP and apharmaceutically acceptable excipient. In certain embodiments thecomposition may also contain, buffers, carriers, stabilizers,preservatives and/or bulking agents, to render the composition suitablefor ocular administration to a patient to achieve a desired effect orresult. In certain embodiments, the pharmaceutical composition comprisesone or more permeability enhancers that permit a non-naturally occurringVEGF-A-binding CKP to penetrate the cornea. Examples of suchpermeability enhancers include, e.g., surfactants, bile acids, chelatingagents, preservatives, cyclodextrins (i.e., cylindrical oligonucleotideswith a hydrophilic outer surface and a lipophilic inner surface thatform complexes with lipophilic drugs), etc. Such permeability enhancersincrease chemical stability and bioavailability and decrease localirritation. In certain embodiments, a pharmaceutical compositionprovided herein additionally comprises agents that increase theabsorption and distribution of non-naturally occurring VEGF-A-bindingCKP in various ocular compartments. In certain embodiments, apharmaceutical composition provided herein comprises a cross-linkedpolyacrylic acid, which can enhance ocular bioavailability by virtue ofits mucoadhesive properties. In certain embodiments, a pharmaceuticalcomposition provided herein comprises a bioadhesive polymer.

In certain embodiments, a pharmaceutical composition provided herein isformulated as an in-situ gelling system, e.g., a viscous polymer-basedliquid that exhibits sol-to-gel phase transition on the ocular surfacedue to change in a specific physicochemical parameter (ionic strength,temperature, pH, or solvent exchange) when the composition comes intocontact with tear fluid. In certain embodiments, a pharmaceuticalcomposition provided herein is formulated as an eye spray. In certainembodiments, a pharmaceutical composition provided is formulated asliposomes. In certain embodiments, a pharmaceutical composition providedherein is formulated as niosomes (i.e., non-ionic surfactant-basedvesicles containing, e.g., cholesterol as an excipient). In certainembodiments, a pharmaceutical composition provided herein is formulatedas pharmacosomes (i.e., vesicles formed by amphiphilic drugs). Incertain embodiments, a pharmaceutical composition provided herein isformulated as a microemulsion. Further details regarding variousophthalmic pharmaceutical formulations are provided in, e.g., Gaikwad etal. (2013) Indo Amer J Pharm Res. 3, 3216-3232; Achouri et al. (2012)Drug Dev Indust Pharm. 39, 1599-1617; Lu (2010) Recent Pat Drug DelivFormul. 4, 49-57; Baranowski et al. (2014) Sci World J.doi.org/10.1155/2014/861904; Lang (1995) Adv Drug Deliv Rev. 16, 39-43;Short (2008) Toxicologic Path. 36, 49-62; and others.

In certain embodiments, a pharmaceutical composition comprisingnon-naturally occurring VEGF-A-binding CKP described herein is stable atroom temperature (such as at about 20-25° C.) for about 0.5 weeks, about1.0 weeks, about 1.5 weeks, about 2.0 weeks, about 2.5 weeks, 3.5 weeks,about 4.0 weeks, about 1 month, about 2 months about 3 months, about 4months about 5 months, about 6 months, or greater than 6 months,including any range in between these values. In certain embodiments, apharmaceutical composition comprising non-naturally occurringVEGF-A-binding CKP described herein is stable under acceleratedconditions (such as storage at about 37° C.) for about 0.5 weeks, about1.0 weeks, about 1.5 weeks, about 2.0 weeks, about 2.5 weeks, 3.5 weeks,about 4.0 weeks, about 1 month, about 2 months about 3 months, about 4months about 5 months, about 6 months, or greater than 6 months,including any range in between these values.

Methods of Treatment Using Non-Naturally Occurring Cystine Knot Peptides(CKPs) that Bind Vascular Endothelial Growth Factor A (VEGF-A)

Vascular endothelial growth factor (VEGF-A), a dimeric glycoprotein ofapproximately 40 kDa, is a potent, endothelial cell mitogen thatstimulates proliferation, migration and tube formation leading toangiogenic growth of new blood vessels and increased vascularpermeability. Low oxygen conditions in the retina or cornea induce theexpression of vascular endothelial growth factor (VEGF-A), and theabnormal (such as excessive or otherwise inappropriate) growth of leakyblood vessels contributes to the pathology of several debilitatingocular diseases including, e.g., diabetic blindness, retinopathies,primarily diabetic retinopathy, age-related macular degeneration (AMD),proliferative diabetic retinopathy (PDR), retinopathy of prematurity(ROP), choroidal neovascularization (CNV), diabetic macular edema,pathological myopia, von Rippel-Lindau disease, histoplasmosis of theeye, retinal vein occlusion (both branched retinal vein occlusion (BRVO)and central retinal vein occlusion (CRVO), corneal neovascularization,retinal neovascularization and rubeosis. The VEGF-A-induced formation ofnew blood vessels is detrimental, and retinal, intertrabecular orcorneal neovascularization can ultimately lead to vision loss.

In certain embodiments, provided herein is a method of treating anocular disease or disorder in a subject comprising administering to thesubject an effective amount of a non-naturally occurring VEGF-A-bindingCKP described herein or a composition (such as a pharmaceuticalcomposition) comprising a non-naturally occurring VEGF-A-binding CKPdescribed herein. In certain embodiment, provided are compositions (suchas pharmaceutical compositions) comprising a non-naturally occurringVEGF-A-binding CKP described herein for use in treating an oculardisease or disorder in a subject. In certain embodiments, provided isthe use of a non-naturally occurring VEGF-A-binding CKP described herein(or composition comprising such non-naturally occurring CKP) in themanufacture of a medicament for the treatment of an ocular disease ordisorder in a subject.

In certain embodiments, the subject to be treated is a mammal (e.g.,human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog,cat, etc.). In certain embodiments, the subject is a human. In certainembodiments, the subject is a clinical patient, a clinical trialvolunteer, an experimental animal, etc. In certain embodiments, thesubject is suspected of having or at risk for having an ocular diseaseor disorder characterized by abnormal angiogenesis and/or abnormalvascular permeability (such as those described herein). In certainembodiments, the subject has been diagnosed with an ocular disease ordisorder characterized by abnormal angiogenesis and/or abnormal vascularpermeability (such as those described herein).

In certain embodiments, the ocular disease or disorder is an ocularvascular proliferative disease, such as an ocular vascular proliferativedisease selected from the group consisting of diabetic blindness,retinopathies, primarily diabetic retinopathy, age-related maculardegeneration (AMD), proliferative diabetic retinopathy (PDR),retinopathy of prematurity (ROP), choroidal neovascularization (CNV),diabetic macular edema, pathological myopia, von Rippel-Lindau disease,histoplasmosis of the eye, retinal vein occlusion (both branched retinalvein occlusion (BRVO) and central retinal vein occlusion (CRVO), cornealneovascularization, retinal neovascularization, and rubeosis. In certainembodiments, the corneal neovascularization results infection of theeye, inflammation in the eye, trauma to the eye (including chemicalburns), or loss of the limbal stern cell barrier. In certainembodiments, the corneal neovascularization results from herpetickeratitis, trachoma, or onchocerciasis.

In certain embodiments, the effective amount of the non-naturallyoccurring VEGF-A-binding CKP described herein (or composition comprisingsuch non-naturally occurring VEGF-A-binding CKP described herein) isadministered directly to the eye of the subject (such as intravitreallyor topically), as described in further detail elsewhere herein.

In certain embodiments, the non-naturally occurring VEGF-A-binding CKPdescribed herein (or composition comprising such non-naturally occurringCKP) is administered in combination with a second agent. For patients inwhom the ocular disease or disorder is triggered by an inflammatoryresponse, combination therapy with an anti-inflammatory agent can beconsidered. For example, the combined use of steroids and anon-naturally occurring VEGF-A-binding CKP described herein (orcomposition comprising such non-naturally CKP) to reduce inflammationand prevent formation of new blood vessels, respectively, may beparticularly advantageous in patients with, e.g., cornealneovascularization. Patients who suffer from an ocular disease ordisorder secondary to bacterial, viral, fungal or acanthamoebalinfection may benefit from administration of a non-naturally occurringVEGF-A-binding CKP described herein (or composition comprising suchnon-naturally occurring CKP) in combination with an antimicrobial agentand optionally an anti-inflammatory agent. Patients with corneal stromalblood vessels as a result of an ocular disease or disorder are at asignificant risk for immune rejection after corneal transplantation.Administration of a non-naturally occurring VEGF-A-binding CKP describedherein (or composition comprising such non-naturally occurring CKP)prior to (and optionally also subsequent to) corneal transplantationtherefore may be particularly beneficial to patients with cornealstromal blood vessels as successful reduction of corneal vascularizationwill reduce the risk of graft rejection. In certain embodiments, thenon-naturally occurring VEGF-A-binding CKP described herein (orcomposition comprising such non-naturally occurring CKP) is administeredin combination with a second anti-angiogenic agent. In certainembodiments, the non-naturally occurring VEGF-A-binding CKP describedherein (or composition comprising such non-naturally occurring CKP) isadministered in combination with a matrix metalloprotease (MMP)inhibitor.

In certain embodiments, the non-naturally occurring VEGF-A-binding CKPdescribed herein (or composition comprising such non-naturally occurringCKP) is administered in combination with a second therapy. In certainembodiments, the second therapy is laser photocoagulation therapy (LPT).LPT uses laser light to cause controlled damage of the retina to producea beneficial therapeutic effect. Small bursts of laser light can sealleaky blood vessels, destroy abnormal blood vessels, seal retinal tears,or destroy abnormal tissue in the back of the eye. It is quick,non-invasive, and usually requires no anesthesia other than ananesthetic eye drop. LPT techniques and apparatuses are readilyavailable to ophthalmologists (see Lock et al. (2010) Med J Malaysia65:88-94). Additional details regarding LPT can be found in, e.g., WO2014/033184.

In certain embodiments, the second therapy is photodynamic therapy(PDT). PDT uses a light-activated molecule to cause localized damage toneovascular endothelium, resulting in vessel occlusion. Light isdelivered to the retina as a single circular spot via a fiber opticcable and a slit lamp, using a suitable ophthalmic magnification lens(laser treatment). The light-activated compound is injected into thecirculation prior to the laser treatment, and damage is inflicted byphotoactivation of the compound in the area afflicted byneovascularization. One commonly used light-activated compound isverteporfin (Visudyne®). Verteporfin is transported in the plasmaprimarily by lipoproteins. Once verteporfin is activated by light in thepresence of oxygen, highly reactive, short-lived singlet oxygen andreactive oxygen radicals are generated which damages the endotheliumsurrounding blood vessels. Damaged endothelium is known to releaseprocoagulant and vasoactive factors through the lipo-oxygenase(leukotriene) and cyclooxygenase (eicosanoids such as thromboxane)pathways, resulting in platelet aggregation, fibrin clot formation andvasoconstriction. Verteporfin appears to somewhat preferentiallyaccumulate in neovasculature. The wavelength of the laser used forphotoactivation of the light-activated compound may vary depending onthe specific light-activated compound used. Additional details regardingPDT can be found in, e.g., WO 2014/033184.

In certain embodiments, the second therapy is diathermy and cautery,wherein vessels are occluded either by application of a coagulatingcurrent through a unipolar diathermy unit or by thermal cautery using anelectrolysis needle inserted into feeder vessels at the limbus.

Administration

In certain embodiments the non-naturally occurring VEGF-A-binding CKP(or composition comprising such non-naturally occurring CKP) isadministered, e.g., via injection, e.g., subconjunctival injection,intracorneal injection, or intravitreal injection. Administration inaqueous form is usual, with a typical volume of 20-150 μl e.g. 40-60 μl,or 50 μl. Injection can be via a 30-gauge×½-inch (12.7 mm) needle. Incertain embodiments, the non-naturally occurring VEGF-A-binding CKP (orcomposition comprising such non-naturally occurring CKP) is provided ina pre-filled sterile syringe ready for administration. In certainembodiments, the syringe has low silicone content or is silicone free.The syringe may be made of glass. Using a pre-filled syringe fordelivery has the advantage that any contamination of the sterileantagonist solution prior to administration can be avoided. Pre-filledsyringes also provide easier handling for the administeringophthalmologist. See, e.g., WO 2014/033184, Fagan et al. (2013) Clin ExpOphthalmol. 41, 500-507; Avery et al. (2014) Retina. 34 Suppl 12,S1-S18; and Doshi et al. (2015) Seminar Ophthalmol. 26, 104-113 forfurther details regarding intravitreal administration.

In certain embodiments, the non-naturally occurring VEGF-A-binding CKP(or composition comprising such non-naturally occurring CKP) isadministered topically, e.g. in form of eye drops. Additional detailsregarding topical drug delivery to the eye are found in, e.g., Loftssonet al. (2012) Acta Ophthalmologica. 90, 603-608; Patel et al. (2013)World J. Pharmacol. 2, 47-64; Freeman et al. (2009) Exp Rev Ophthalmol.4, 59-64; and Boddu et al. (2014) Recent Patents on Drug Delivery andFormulation. 8, 27-36.

In certain embodiments, an intravitreal device is used to continuouslydeliver the non-naturally occurring VEGF-A-binding CKP (or compositioncomprising such non-naturally occurring CKP) into the eye. In certainembodiments, the non-naturally occurring VEGF-A-binding CKP (orcomposition comprising such non-naturally occurring CKP) is administeredvia ocular insert (including, but not limited to, e.g., Ocuserts,Lactisers, Soluble Ocular Drug Inserts (SODIs), Minidiscs, contactlenses, films, filter paper strips, artificial tear inserts, andcollagen shields). See, e.g., Gaikwad et al. (2013) Indo Amer J PharmRes. 3, 3216-3232). In certain embodiments, the non-naturally occurringVEGF-A-binding CKP (or composition comprising such non-naturallyoccurring CKP) is administered as a slow-release depot, an ocularplug/reservoir, an ocular implant (such as a scleral or vitrealimplant). Various scleral and intravitreal delivery systems are known inthe art. These delivery systems are typically non-biodegradable, and maybe active or passive. For example, WO 2010/088548 describes a deliverysystem having a rigid body using passive diffusion to deliver atherapeutic agent. WO 2002/100318 discloses a delivery system having aflexible body that allows active administration via a pressuredifferential. Alternatively, active delivery can be achieved byimplantable miniature pumps. An example for an intravitreal deliverysystem using a miniature pump to deliver a therapeutic agent is theOphthalmic MicroPump System™ marketed by Replenish, Inc. which can beprogrammed to deliver a set amount of a therapeutic agent for apre-determined number of times. In certain embodiments, thenon-naturally occurring VEGF-A-binding CKP (or composition comprisingsuch non-naturally occurring CKP) is encased in a small capsule-likecontainer (e.g., a silicone elastomer cup). The container is usuallyimplanted in the eye above the iris. The container comprises a releaseopening. Release of the non-naturally occurring VEGF-A-binding CKP (orcomposition comprising such non-naturally occurring CKP) may becontrolled by a membrane positioned between the non-naturally occurringVEGF-A-binding CKP (or composition comprising such non-naturallyoccurring CKP) and the opening, or by means of a miniature pumpconnected to the container. Alternatively, the non-naturally occurringVEGF-A-binding CKP (or composition comprising such non-naturallyoccurring CKP) may be deposited in a slow-release matrix that preventsrapid diffusion of the antagonist out of the container. Preferably, theintravitreal device is designed to release the non-naturally occurringVEGF-A-binding CKP (or composition comprising such non-naturallyoccurring CKP) at an initial rate that is higher in the first month. Therelease rate slowly decreases, e.g., over the course of the first monthafter implantation, to a rate that is about 50% less than the initialrate. The container may have a size that is sufficient to hold a supplyof the non-naturally occurring VEGF-A-binding CKP (or compositioncomprising such non-naturally occurring CKP) that lasts for about fourto six months. Since a reduced dose of the non-naturally occurringVEGF-A-binding CKP (or composition comprising such non-naturallyoccurring CKP) may be sufficient for effective treatment whenadministration is continuous, the supply in the container may last forone year or longer, preferably about two years, more preferably aboutthree years. Because only a small surgery is required to implant adelivery system and intravitreal injections are avoided, patientcompliance issues with repeated intravitreal injections can be avoided.Intravitreal concentrations of the non-naturally occurringVEGF-A-binding CKP (or composition comprising such non-naturallyoccurring CKP) are reduced, and therefore the potential risk ofside-effects from the non-naturally occurring VEGF-A-binding CKP (orcomposition comprising such non-naturally occurring CKP) entering thecirculation is decreased.

In certain embodiments, the non-naturally occurring VEGF-A-binding CKP(or composition comprising such non-naturally occurring CKP) isadministered via iontophoresis. Iontophoresis is a noninvasive techniquein which a small electric current is applied to enhance ionized drugpenetration into tissue (see, e.g., Myles et al. (2005) Adv Drug DelivRev 57, 2063-79 and Eljarrat-Binstock et al. (2006) J Controlled Release110, 479-89). The drug is applied with an electrode carrying the samecharge as the drug, and the ground electrode, which is of the oppositecharge, is placed elsewhere on the body to complete the circuit. Thedrug serves as the conductor of the current through the tissue.

Additional details regarding administration of drug to the eye areprovided in, e.g., Kuno et al. (2011) Polymers 3, 193-221; Short (2008)Toxicologic Path. 36, 49-62; Ghateet al. (2006) Expert Opin Drug Deliv3, 275-87; Davis et al. (2004) Curr Opin Mol Therap 6, 195-205; Gaudanaet al. (2010) AAPS J. 12, 348-360; and others.

Slow Release/Long Acting Delivery Formulations

In certain embodiments, the non-naturally occurring VEGF-A-binding CKP(or composition comprising such non-naturally occurring CKP) is providedas slow-release formulations. Slow-release formulations are typicallyobtained by mixing a therapeutic agent with a biodegradable polymer orencapsulating it into microparticles.

A slow-release formulation in accordance with the invention typicallycomprises the non-naturally occurring VEGF-A-binding CKP (or compositioncomprising such non-naturally occurring CKP), a polymeric carrier, and arelease modifier for modifying a release rate of the non-naturallyoccurring VEGF-A-binding CKP (or composition comprising suchnon-naturally occurring CKP) from the polymeric carrier. By varying themanufacture conditions of polymer-based delivery compositions, therelease kinetic properties of the resulting compositions can bemodulated. The polymeric carrier usually comprises one or morebiodegradable polymers or copolymers or combinations thereof. Forexample, the polymeric carrier may be selected from poly-lactic acid(PLA), poly-glycolic acid (PGA), polylactide-co-glycolide (PLGA),polyesters, poly (orthoester), poly(phosphazine), poly (phosphateester), polycaprolactones, or a combination thereof.

In certain embodiments the polymeric carrier is PLGA. The releasemodifier is typically a long chain fatty alcohol, preferably comprisingfrom 10 to 40 carbon atoms. Commonly used release modifiers includecapryl alcohol, pelargonic alcohol, capric alcohol, lauryl alcohol,myristyl alcohol, cetyl alcohol, palmitoleyl alcohol, stearyl alcohol,isostearyl alcohol, elaidyl alcohol, oleyl alcohol, linoleyl alcohol,polyunsaturated elaidolinoleyl alcohol, polyunsaturated linolenylalcohol, elaidolinolenyl alcohol, polyunsaturated ricinoleyl alcohol,arachidyl alcohol, behenyl alcohol, erucyl alcohol, lignoceryl alcohol,ceryl alcohol, montanyl alcohol, cluytyl alcohol, myricyl alcohol,melissyl alcohol, and geddyl alcohol.

In certain embodiments, the non-naturally occurring VEGF-A-binding CKP(or composition comprising such non-naturally occurring CKP) isincorporated into a microsphere-based sustained release composition. Incertain embodiments, the microspheres are prepared from PLGA. The amountof the non-naturally occurring VEGF-A-binding CKP (or compositioncomprising such non-naturally occurring CKP) incorporated in themicrospheres and the release rate of the non-naturally occurringVEGF-A-binding CKP (or composition comprising such non-naturallyoccurring CKP) can be controlled by varying the conditions used forpreparing the microspheres. Processes for producing such slow-releaseformulations are described in US 2005/0281861 and US 2008/0107694.

In certain embodiments, the non-naturally occurring VEGF-A-binding CKP(or composition comprising such non-naturally occurring CKP) isincorporated into a biodegradable implant (such as a microneedle).Matrix implants (such as microneedles) are typically used to treatocular diseases that require a loading dose followed by tapering dosesof the drug during a 1-day to 6-month time period (Davis et al. (2004)Curr Opin Mol Therap 6, 195-205). They are most commonly made from thecopolymers poly-lactic-acid (PLA) and/or poly-lactic-glycolic acid(PLGA), which degrade to water and carbon dioxide. The rate and extentof drug release from the implant can be decreased by altering therelative concentrations of lactide (slow) and glycolide (fast), alteringthe polymer weight ratios, adding additional coats of polymer, or usinghydrophobic, insoluble drugs. The release of drug generally followsfirst-order kinetics with an initial burst of drug release followed by arapid decline in drug levels. Biodegradable implants do not requireremoval, as they dissolve over time (Hsu (2007) Curr Opin Ophthalmol 18,235-9). Biodegradable implants also allow flexibility in dose andtreatment from short duration (weeks) to longer duration (months to ayear), depending on the polymer PLA/PLGA ratio, which is another benefitin tailoring drug delivery to disease progression, because dose andtreatment requirements may change over time. Additional detailsregarding the manufacture and implantation of biodegradable implants(such as PLGA or PLA implants) for the ocular administration areprovided in, e.g., WO 2006/093758, US 2006/0182783, WO 2009/026461, US2008/0181929, US 2009/0263460, US 2010/0015158, US 2011/0207653, and US2014/0154321. Additional details regarding microneedles for ocular drugdelivery are provided in, e.g., Donnelly et al. (2010) Drug Deliv 14,187-207; U.S. Pat. No. 7,918,814, Yavux et al. (2013) Sci World J.doi.org/10.1155/2013/732340, and elsewhere.

Articles of Manufacture and Kits

In certain embodiments, provided is an article of manufacture containinga non-naturally occurring VEGF-A-binding CKP described herein andmaterials useful for the treatment of an ocular disease or disorder(such as an ocular vascular proliferative disease or ocular disordercharacterized by excessive angiogenesis). The article of manufacture cancomprise a container and a label or package insert on or associated withthe container. Suitable containers include, for example, bottles, vials,syringes, etc. The containers may be formed from a variety of materialssuch as glass or plastic. In certain embodiments, the container holds acomposition which is effective for treating the ocular disease ordisorder (such as an ocular vascular proliferative disease or oculardisorder characterized by excessive angiogenesis) and may have acomplete set of items needed to implant a slow release ocular orintraocular drug delivery system, including, but not limited to,injection devices, topical and injectable medications, surgicalinstruments, sutures and suturing needles, and eye covers. In certainembodiments, the container fold sterile unit-dose packages. At least oneactive agent in the composition is non-naturally occurringVEGF-A-binding CKP described herein. The label or package insertindicates that the composition is used for treating an ocular disease ordisorder (such as an ocular vascular proliferative disease or oculardisorder characterized by excessive angiogenesis). The label or packageinsert will further comprise instructions for administering thenon-naturally occurring VEGF-A-binding CKP (or composition comprisingsuch non-naturally occurring CKP) to the patient. Articles ofmanufacture and kits comprising combinatorial therapies described hereinare also contemplated.

Package insert refers to instructions customarily included in commercialpackages of therapeutic products that contain information about theindications, usage, dosage, administration, contraindications and/orwarnings concerning the use of such therapeutic products. In certainembodiments, the package insert indicates that the compositioncomprising the non-naturally occurring VEGF-A-binding CKP is used fortreating an ocular disease or disorder (such as an ocular vascularproliferative disease or ocular disorder characterized by excessiveangiogenesis described herein).

Kits are also provided that are useful for various purposes, e.g., forisolation or detection VEGF-A, optionally in combination with thearticles of manufacture. For isolation and purification of VEGF-A, thekit can contain non-naturally occurring VEGF-A-binding CKP describedherein coupled to beads (e.g., sepharose beads). Kits can be providedwhich contain the non-naturally occurring VEGF-A-binding CKP describedherein for detection and quantitation of VEGF-A in vitro, e.g. in anELISA or blot. As with the article of manufacture, the kit comprises acontainer and a label or package insert on or associated with thecontainer. For example, the container holds a composition comprising atleast one non-naturally occurring VEGF-A-binding CKP described herein.Additional containers may be included that contain, e.g., diluents andbuffers, control antibodies, etc. The label or package insert mayprovide a description of the composition as well as instructions for theintended in vitro or diagnostic use.

Examples Example 1: Materials and Methods for Examples 2-3

Display of EETI-II on M13 Phage.

EETI-II was displayed on the surface of M13 bacteriophage by modifying apreviously described phagemid pS2202b (Skelton, N. J., Koehler, M. F.,Zobel, K., Wong, W. L., Yeh, S., Pisabarro, M. T., Yin, J. P., Lasky, L.A., and Sidhu, S. S. (2003) Origins of PDZ domain ligand specificity.Structure determination and mutagenesis of the Erbin PDZ domain. J BiolChem 278, 7645-7654). Standard molecular biology techniques were used toreplace the fragment of pS2202d encoding Erbin PDZ domain with a DNAfragment encoding for EETI-II. The resulting phagemid (p8EETI-II)contained an open reading frame that encoded for the maltose bindingprotein secretion signal, followed by a gD tag and EETI-II and endingwith M13 major coat protein p8. E. Coli harboring p8EETI-II wereco-infected with M13-KO7 helper phage and cultures were grown in 30 ml2YT medium supplemented with 50 μg/ml Carbenecillin and 25 μg/mlKanamycin at 30° C. overnight. The propagated phage was purifiedaccording to the standard protocol (Tonikian, R., Zhang, Y., Boone, C.,and Sidhu, S. S. (2007) Identifying specificity profiles for peptiderecognition modules from phage-displayed peptide libraries. Nat Protoc2, 1368-1386) and re-suspended in 1 ml PBT buffer (PBS, 0.5% BSA and0.1% TWEEN®20), resulting in the production of phage particles thatencapsulated p8EETI-II DNA and displayed EETI-II. The display level wasanalyzed using a phage ELISA.

Library Construction and Sorting.

The EETI-II libraries were constructed following Kunkel mutagenesismethod (Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Rapidand efficient site-specific mutagenesis without phenotypic selection.Methods Enzymol 154, 367-382). Three libraries were constructed: Library1, in which loop 1 (3-8) was randomized with the degenerated codonencoding all natural amino acids except Cys at 6, 8 or 10 amino acids inlength; or Library 2, in which loop 5 (22-26) was randomized with thesame set of degenerated codon with fixed length of 5 amino acids; orLibrary 3, in which both loop 1 were randomized with 6, 8, and 10 aminoacids and loop 5 with 5 amino acids simultaneously with degeneratedcodon encoding for 19 amino acids. Oligonucleotides for mutagenesis weresynthesized using custom mixes of trimer phosphoramidites encoding for19 amino acids at equimolar concentration. (Glen Research, Sterling,Va.). The stop template is the single strand DNA of p8EETI-II containingthree stop codons in region of 3-26 and was used to construct all threelibraries. The pool of three libraries contained ˜3×10¹⁰ unique membersand was cycled through rounds of binding selection against hVEGF (8-109)captured on plate for four rounds following the standard protocol(Tonikian, R., Zhang, Y., Boone, C., and Sidhu, S. S. (2007) Identifyingspecificity profiles for peptide recognition modules fromphage-displayed peptide libraries. Nat Protoc 2, 1368-1386) with thevariation that, 25 ug/ml of hVEGF(8-109) was used to coat the plate andeluted phage were propagated by growing the overnight culture at 30° C.

Spot Phage ELISA.

After four rounds of binding selection, individual phage clones werepicked and inoculated into 450 μl 2YT media containing 50 μg/mlCarbenecillin and M13-KO7 helper phage in 96-well blocks, which weregrown at 37° C. overnight. The supernatant was analyzed with spot phageELISA as follows: hVEGF(8-109) or BSA were coated on 384-well MAXISORP™immunoplates and phage supernatant diluted (1:3) with PBT buffer wasadded to the wells. The plates were washed and bound phage was detectedwith anti-M13-HRP followed by TMB substrate. In these assays, phagebinding to BSA alone was tested in parallel to assess backgroundbinding. Clones whose binding signals for hVEGF-A (8-109) were more than3 times higher than to BSA (background) were considered positive.Positive clones were subjected to DNA sequence analysis.

Crystallography.

To form a stable complex, VEGF-A was concentrated to 7 mg/ml andincubated with a 6-fold molar excess of VEGF_CKP9.54.90 variant.VEGFA/VEGF_CKP9.54.90 crystals of the primitive monoclinic space groupP12₁1 were grown at 19° C. by the hanging-drop vapor diffusion methodusing a drop ratio of 2:1 protein: reservoir solution. Reservoirsolution contained 100 mM HEPES pH 7.4 and 26% PEG 3350. Crystals werecryoprotected in reservoir solution supplemented with 25% PEG 200 andflash-frozen in liquid nitrogen prior to data collection.

Data Collection and Structure Determination.

X-ray diffraction data were collected to 1.64 Å at beamline 21IDF at theAdvanced Photon Source. Data were processed using iMosflm. The structurewas solved by molecular replacement using Phaser in Phenix with thepreviously published apo VEGF-A structure (PDB: 1VPF) as a search modeland one VEGF-A dimer in the asymmetric unit. Clear F_(o)-F_(c) densitywas present for the VEGF_CKP9.54.90 variant, so the structure of thisvariant was built into the density manually using Coot and thensubjected to iterative rounds of refinement and rebuilding using Phenixand Coot.

KDR-CHO VEGF Assay to Determine Cellular IC₅₀

KDR-CHO cells (CHO cells stably transfected with gD tagged-KDR) weregrown in cell growth medium (DMEM/Ham's F-12, 10% diafiltered FBS (GIBCOcatalog no. 26400), 25 mM HEPES, 2 mM L-GLUTAMAX™). For VEGF stimulationassay, 5×10⁴ cells/well were plated in 100 μl of cell plating medium(DMEM/Ham's F-12, 0.2% BSA, 0.25% diafiltered FBS, 25 mM HEPES, 2 mML-GLUTAMAX™) in 96-well tissue culture plate and incubated at 37° C.overnight. The medium was replaced with 100 μl of serum-free cellstimulation medium (DMEM/Ham's F-12, 0.5% BSA, 25 mM HEPES) and cellswere incubated at 37° C. for 2 hr. One hour before stimulation, themedium was replaced with 50 μl of serum-free cell stimulation medium.Concurrently, VEGF (50 ng/ml for hVEGF, 100 ng/ml for mVEGF and rVEGF)was pre-incubated with titrated amount of CKP or anti-VEGF in 50 μl ofserum-free cell stimulation medium at 37° C. for 1 hour and added to thecells. The cells were stimulated for 15 min at 37 C and the medium isremoved. The cells were lysed with 130 μl of ice-cold cell lysis buffer(150 mM NaCl, 50 mM HEPES, 0.5% Triton-X 100, HALT protease andphosphatase inhibitor cocktail (ThermoFisher Scientific, Inc. catalogno. 78444), 5 mM EDTA). VEGF mediated Tyr phosphorylation of KDR wasdetermined by ELISA-based assay. Briefly, MAXISORP™ 96 well plates(ThermoFisher Scientific, Inc. catalog no. 439454) were coated with 100μl of anti-gD antibody diluted in PBS (1 μg/ml) at 4° C. overnight andwashed three times with washing buffer (PBS, 0.05% TWEEN®20, pH 7.4).The plates were blocked with 300 μl of blocking buffer (PBS, 0.5% BSA)at room temperature for 1 hour followed by washing three times withwashing buffer. The above KDR-CHO cell lysate (100 μl) was added to eachwell and incubated at room temperature for 2 hours. The plates werewashed four times with washing buffer followed by incubation with 100 μlof 0.5 μg/ml biotin-conjugated anti-phosphotyrosine (clone 4G10,Millipore catalog no. 16-103) in blocking buffer at room temperature for2 hours. After washing four times, the plates were incubated with 100 μlof HRP-conjugated streptavidin in blocking buffer at room temperaturefor 30 min. After washing four times, the plates were developed with 100μl of TMB substrate (BD Biosciences) at room temperature for 20-30 minand stopped by addition of 50 μl of H₂SO₄ solution. The optical densityof each well was determined using a microplate reader set to 450 nm.

Competition ELISAs

Binding specificity of each peptide was established by competitionELISA. First, binding of each growth factor to their correspondingreceptor in a plate-ELISA format was confirmed by coating VEGF-A,VEGF-B, VEGF-C, VEGF-D, PlGf-2, NGF, EGF, PDGF-ββ, or IGF-1 at 2 or 5μg/mL in MAXISORP™ plates overnight at 4° C. in PBS. After blocking withblock buffer (PBS with 0.5% BSA and 0.05% TWEEN®20) for 2 hours at roomtemperature, the receptor-Fc fusions or biotinylated receptors wereserially diluted using assay buffer (PBS with 0.5% BSA and 0.05%TWEEN®20) and incubated for 1 hour at room temperature. Amount of boundreceptor-Fc or biotinylated receptor was detected by incubating withanti-human-Fc-HRP (Life Technologies) or high affinity streptavidin-HRP(ThermoFisher Scientific, Inc.) respectively for 30 min. CompetitionELISA was conducted in an identical fashion as described above exceptafter blocking, a mixture of serially diluted peptide containing aconstant concentration of receptor-Fc fusion or biotinylated receptor(concentration of receptor was set to EC₆₀) was added and incubated for1 hour. All recombinant human proteins and antibodies were purchasedfrom R &D Systems (Minneapolis, Minn.).

SPR Binding Assays.

Binding kinetics and affinities of inhibitors of VEGF-A were assessedusing surface plasmon resonance technology on a BIACORE™ 3000 instrument(GE Healthcare) at 37° C. using HBS-EP buffer (10 mM HEPES pH 7.4, 150mM NaCl, 3 mM EDTA and 0.005% v/v surfactant P20) containing 0.1% DMSO(v/v). Depending on the format of the assay either a streptavidin sensor(SA) or a dextran-coated (CM5) sensor was utilized as described below.

For use with the SA sensor, VEGF-A was first biotinylated (no more than2 biotin/VEGF-A) by incubating the protein with EZ-link NHS-PEG4-Biotin(Pierce) in a 1:1.5 molar ratio respectively, in PBS for 2 hours on ice.Reaction was then quenched by addition of 10 molar excess of Glycine pH8.0 and the sample was buffer exchanged into PBS using an Amicon 0.5 mL3000 MWCO ultra-centrifugal filters (EMD Millipore). The biotinylationstate of the protein was verified by LC-MS analysis. Biotinylated VEGF-Awas then captured on the surface until a resonance unit (RU) signal ofabout ˜400. For immobilization of VEGF-A on CMS sensor, the surface wasfirst activated with a mixture ofN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride andN-hydroxysuccinimide (EDC/NHS) according to the supplier instructions.VEGF-A was then diluted into coupling buffer (0.1 M Acetate Buffer, pH5.0) and injected until the signal reached about ˜400 RU followed by awash with 1 M ethanolamine pH 8.0 to quench remaining activated sites.

Following the capture step, a series of the peptide concentrations wereprepared in HBS-EP buffer with matching DMSO concentrations to 0.1% andinjected at a flow rate of 80 μL/min. The resulting sensorgrams werethen analyzed using a 1:1 binding model to obtain kinetic data andaffinities using Scrubber 2.0 (BioLogic Software).

CKP Synthesis and Folding.

Linear precursor LRP6 peptides were dissolved into DMSO (0.5 mg/mL) into0.1M ammonium bicarbonate (pH 9), 1 mM reduced glutathione in 50% DMSOand incubated while shaking at room temperature for 24 h. Folded CKPswere purified by RP-HPLC on a C18 column and then collected fractionswere analyzed by mass spectrometry, pooled and lyophilized prior to use.

Cell Culture and Transfection.

HEK293 cells stably transfected with a firefly luciferase Wnt reporter(Gong et al. (2010) PLoS ONE 5, 9: e12682). and pRL-SV40 Renillaluciferase (Promega) were grown to 90% confluence in DMEM:F12 (50:50)supplemented with 10% FBS, 2 mM GLUTAMAX™ and 40 μg/ml hygromycin. Cellswere incubated in a 5% CO₂ humidified incubator at 37° C. for 24 h.Following the incubation, the cells were trypsinized (0.05% Gibco15400-54 in PBS) then diluted to 4×10⁵ cells/ml in DMEM:F12 (50:50)supplemented with 10% FBS, 2 mM GLUTAMAX™. 20,000 cells were loaded intoindividual wells of white microtest 96-well optilux plates (catalog no.353947) and incubated for ˜24 h. Each well was transfected using FUGENE®HD with Wnt1-pCDNA3.2 (5 ng/well) or Wnt3a-pCDNA3.2 (25 ng/well) thengrown for 24 h. All LRP6-binding variants were diluted in DMSO and addedto cells at a final DMSO concentration of 1% at peptide concentrationsof 0, 0.1, 0.1, 1.0, 10, and 100 μM for 6 hours. For stimulation withrecombinant Wnt3a (5036-WN-010/CF, R&D Systems) was diluted in PBS to 50ng/mL and added to the incubation media with the indicated CKP.

Luciferase response in all assays was then measured with Promega'sDUAL-GLO® kit according to the manufacturer's instructions, except usinghalf the volume of each reagent. Firefly luminescence and Renillaluminescence were measured on a Perkin Elmer ENVISION™ MultilabelReader. The ratios of firefly luminescence: Renilla luminescence werecalculated and normalized to the ratio in control cells expressing ortreated with the indicated Wnt protein. Inhibitory constants werecalculated using normalized data in Prism Graphpad using the using thelog(inhibitor) vs. normalized response−variable slope Y=100/(1+10̂((LogIC₅₀−X)*HillSlope)). Statistical significance was determined using theHolm-Sidak method, with alpha=5.000%. Computations assume that all rowsare sample from populations with the same scatter (SD) and IC₅₀ wereidentified as significantly different using the Extra sum-of-squares Ftest where P<0.05 when significant.

Example 2A: Generation of Non-Naturally Occurring EETI-II Variants thatBind VEGF-A

EETI-II (FIG. 1) was chosen as a scaffold for display on the surface ofM13 bacteriophage. EETI-II was fused to the N terminus of M13 major coatprotein p8. Furthermore, a gD-tag was engineered N terminal of EETI-IIsequence in order to verify display levels. Three peptide phagelibraries were generated based on the EETI-II framework as follows:library I in which loop 1 amino acid residues were randomized and theloop length was varied (6, 8, 10 residues); library II in which loop 5amino acid residues were randomized and the native loop length wasfixed; and library III in which both loops 1 and 5 were randomized inamino acid content simultaneously and loop 1 length was varied from 6 to10 amino acid residues while loop 5 length was fixed. Altogether, thethree libraries contained 3×10¹⁰ unique members and were cycled throughrounds of selection against VEGF-A.

Panning against VEGF-A generated thirteen unique variants that bound tohVEGF₈₋₁₀₉ (see Table 19). These variants contained variations in aminoacid composition in loop 1 or both loops 1 and 5 simultaneously. Also, anumber of variants had a longer loop compared to the native loop presentin EETI-II. A conserved YXS motif was also apparent in loop 5. Wegenerated soluble folded cystine-knot peptides that correspond to sevenof these unique variants, and they all demonstrated binding to hVEGF-Ain a phage competition ELISA (Table 19). Moreover, we assessed some ofthe variants in a cellular assay and they demonstrated cross-speciesinhibition of human, mouse and rat VEGF-A activity with IC₅₀ in low μM.

TABLE 19 EETI-II-based binders against hVEGF-A VARIANT LOOP 1 LOOP 5 nELISA S/N* EETI-II PRILMR GPNGF 0.01   1.11 (SEQ ID NO: 92)(SEQ ID NO: 15) VEGF_CKP1 ETDWYPHQID GPNGF  2 0.9  16.9 (SEQ ID NO:(SEQ ID NO: 15) 225) VEGF_CKP2 GETVFEQFLW GPNGF  2 3.2  48.1 (SEQ ID NO:(SEQ ID NO: 15) 226) VEGF_CKP3 HMMYDY EMYDA  2 3.1  42.9 (SEQ ID NO:(SEQ ID NO: 227) 235) VEGF_CKP4 KKWQWWYM YPWTE  5 2.6  35.3 (SEQ ID NO:(SEQ ID NO: 228) 236) VEGF_CKP5 PAIQNWKEHP SWWPSL  2 1.9  28.4(SEQ ID NO: (SEQ ID NO: 229) 237) VEGF_CKP6 PTTRFKQY GPNGF 28 3.5  51.9(SEQ ID NO: 8) (SEQ ID NO: 15) VEGF_CKP7 QDPTFNWALY QMYQS  2 3.4  54.5(SEQ ID NO: 9) (SEQ ID NO: 16) VEGF_CKP8 QLMHPFWG HWYRS  2 3.9  59.4(SEQ ID NO: (SEQ ID NO: 230) 238) VEGF_CKP9 QLMQPFWG HWYQS 11 3.3  36.0(SEQ ID NO: 10) (SEQ ID NO: 17) VEGF_CKP10 RDLDVKWD QYYSS  3 3.2  43.7(SEQ ID NO: 11) (SEQ ID NO: 18) VEGF_CKP11 RTPWEPHDIT GPNGF 16 4.1  57.5(SEQ ID NO: 12) (SEQ ID NO: 15) VEGF_CKP12 TTPWPPHEIM GPNGF 75 3.5  55.2(SEQ ID NO: 13) (SEQ ID NO: 15) VEGF_CKP13 VTPWKPHWIN GPNGF  2 3.5  56.3(SEQ ID NO: 14) (SEQ ID NO: 15) *S/N = signal to noise ratio as comparedto BSA control

TABLE 20 Phage Competition ELISA and cellular inhibitory activities ofsoluble EETI-II-based binders against hVEGF-A Cellular Assay In vitrohVEGF-A mVEGF rVEGF VARIANT IC₅₀ (μM) (25 ng/ml) (50 ng/ml) (50 ng/ml)VEGF_CKP6 3 47 169 711.2 (partial) VEGF_CKP7 12 18 39 7 VEGF_CKP9 0.6 1210 1 VEGF_CKP10 1 45 ND* 18 VEGF_CKP11 80 ND* ND* ND* VEGF_CKP12 60 102243 1220 (partial) VEGF_CKP13 80 ND* ND* ND* *ND= not determined

To further improve the potency of these variants, we followed up onVEGF_CKP7 and VEGF_CKP9. Soft randomization was done on loops 1 and 5within the VEGF-CKP7 framework, resulting in 16 unique variants thatbound to human VEGF-A (see Table 21 below).

TABLE 21 CKP_7 Affinity-matured binders against hVEGF-A VARIANT LOOP 1LOOP 5 n ELISA S/N* VEGF_CKP7 QDPTFNWALY QMYQS 1 0.1  1.3 (SEQ ID NO: 9)(SEQ ID NO: 16) VEGF_CKP7.2 DDPSFDWSVY RMYDS 1 1.2 21.5 (SEQ ID NO:(SEQ ID NO: 287) 292) VEGF_CKP7.8 KNPLFNWALY QLFDS 2 0.5  7.5(SEQ ID NO: SEQ ID NO: 71) 60) VEGF_CKP7.17 QDPTVNWAVY QFYQS 1 0.8 13.4(SEQ ID NO: (SEQ ID NO: 72) 61) VEGF_CKP7.19 QDPTFNWAEY QLYQS 2 0.6 11.1(SEQ ID NO: (SEQ ID NO: 73) 62) VEGF_CKP7.24 WDPTFNWALY QMYDS 2 0.8 13.4(SEQ ID NO: (SEQ ID NO: 76) 288) VEGF_CKP7.35 QDPTFNWAEY QMYQS 3 0.610.6 (SEQ ID NO: (SEQ ID NO: 16) 62) VEGF_CKP7.43 QDPTLNWATY QMYQS 1 0.5 6.3 (SEQ ID NO: (SEQ ID NO: 16) 289) VEGF_CKP7.46 EDPTVDWAQY QMYQS 10.3  4.9 (SEQ ID NO: (SEQ ID NO: 16) 290) VEGF_CKP7.50 QDPSLNWADY QMHQS1 0.8 14.3 (SEQ ID NO: (SEQ ID NO: 74) 63) VEGF_CKP7.54 LDRTLNWALY QMYNS1 0.5  9.3 (SEQ ID NO: (SEQ ID NO: 75) 64) VEGF_CKP7.57 LDPSFNWSLY QMYDS2 1.0 17.4 (SEQ ID NO: (SEQ ID NO: 76) 65) VEGF_CKP7.73 RDLTINWALF QMFNS1 1.2 19.2 (SEQ ID NO: (SEQ ID NO: 66) 274) VEGF_CKP7.78 KDTTFNWGLFQLYQS 1 0.7 11.8 (SEQ ID NO: (SEQ ID NO: 73) 291) VEGF_CKP7.81LDPTVNWALF QHYKT 1 1.1 18.6 (SEQ ID NO: (SEQ ID NO: 77) 67) VEGF_CKP7.88QDPKLNWAVY QLFNS 2 0.5  7.7 (SEQ ID NO: (SEQ ID NO: 78) 68) LRP6_CKP7.89LDPSFDWALY QLYNS 1 0.5  8.1 (SEQ ID NO: (SEQ ID NO: 79) 69) *S/N =signal to noise ratio as compared to BSA control

Soft randomization was done on loops 1 and 5 within the VEGF-CKP9framework, resulting in 16 unique variants that bound to human VEGF-A(see Tables 22-24 below).

TABLE 22 VEGF_CKP9 Loop1 Affinity-Matured Variants Against VEGF-AVARIANT L1 SEQUENCE n ELISA S/N* VEGF_CKP9 QLMQPFWG 0.4  7.4(SEQ ID NO: 10) VEGF_CKP9.L1.2 HLFEPLWG 17 1.6 12.5 (SEQ ID NO: 245)VEGF_CKP9.L1.7 QVMRPFWG  3 1.5  8.9 (SEQ ID NO: 246) VEGF_CKP9.L1.8QVMQPAWG  1 1.2 12.8 (SEQ ID NO: 247) VEGF_CKP9.L1.19 HRLQPLWG  3 1.411.6 (SEQ ID NO: 248) VEGF_CKP9.L1.26 ELLQPSWG  4 1.7 11.9(SEQ ID NO: 249) VEGF_CKP9.L1.57 NPMLPFWG  3 3.7 31.8 (SEQ ID NO: 368)VEGF_CKP9.L1.64 NVLLPLWG  1 2.3 19.9 (SEQ ID NO: 250) VEGF_CKP9.L1.68DIMQPLWG  1 2.0 23.2 (SEQ ID NO: 36) VEGF_CKP9.L1.76 DLMQPLWG  2 2.517.4 (SEQ ID NO: 251) VEGF_CKP9.L1.78 NPMLPLWG  1 3.0 25.1(SEQ ID NO: 252) VEGF_CKP9.L1.79 QVLQPSWG  1 1.2 10.0 (SEQ ID NO: 253)*S/N = signal to noise ratio as compared to BSA control

TABLE 23 VEGF_CKP9 Loop5 Affinity-Matured Variants Against VEGF-AVARIANT L5 SEQUENCE n ELISA S/N* VEGF_CKP9 HWYQS 0.9 11.6(SEQ ID NO: 17) VEGF_CKP9.L5.7 RWYNS 11 1.4 18.9 (SEQ ID NO: 133)VEGF_CKP9.L5.18 HWYQS  1 1.6 20.4 (SEQ ID NO: 17) VEGF_CKP9.L5.43 RWYHS 2 0.9 13.6 (SEQ ID NO: 43) *S/N = signal to noise ratio as compared toBSA control

TABLE 24 VEGF_CKP9 Loop1/Loop5 Affinity-Matured Variants Against VEGF-AVARIANT Loop 1 LOOP 5 n ELISA S/N* VEGF_CKP9 QLMQPFWG HWYQS 0.4  7.4(SEQ ID NO: 10) (SEQ ID NO: 17) VEGF_CKP9.2 DVLQPFWG HWYQS 20 2.4 31.1(SEQ ID NO: 28) (SEQ ID NO: 17) VEGF_CKP9.3 QISQPFWG HFYNS  1 1.6 24.5(SEQ ID NO: 29) (SEQ ID NO: 41) VEGF_CKP9.4 DRMQPLWG LWYKS N/D N/D(SEQ ID NO: 30) (SEQ ID NO: 42) VEGF_CKP9.9 QLLEPMWG HWYNS  1 1.1 19.7(SEQ ID NO: 254) (SEQ ID NO: 46) VEGF_CKP9.14 KLLQPMWG RWYQS  1 2.0 30.1(SEQ ID NO: 255) (SEQ ID NO: 44) VEGF_CKP9.11 DRMQPYWG QWYKS  1 1.1 14.1(SEQ ID NO: 256) (SEQ ID NO: 262) VEGF_CKP9.20 NLMLPFWG RWYHS  1 1.713.3 (SEQ ID NO: 31) (SEQ ID NO: 43) VEGF_CKP9.22 QRTQPFWG RWYQS  1 1.217.2 (SEQ ID NO: 32) (SEQ ID NO: 44) VEGF_CKP9.47 KIMQPLWG LWYDS  1 1.014.6 (SEQ ID NO: 257) (SEQ ID NO: 263) VEGF_CKP9.51 NLMHPFWG HWYQS  11.0 11.0 (SEQ ID NO: 258) (SEQ ID NO: 17) VEGF_CKP9.54 NIMLPFWG QYYQS  12.1 28.4 (SEQ ID NO: 33) (SEQ ID NO: 45) VEGF_CKP9.59 DPMQPFWG RWYQS N/DN/D (SEQ ID NO: 34) (SEQ ID NO: 44) VEGF_CKP9.63 DVMQPYWG HWYNS  1 2.029.7 (SEQ ID NO: 35) (SEQ ID NO: 46) VEGF_CKP9.69 ALLQPLWG RWYNS  1 1.014.3 (SEQ ID NO: 259) (SEQ ID NO: 133) VEGF_CKP9.71 QLLQPLWG RWYQS  11.0 16.5 (SEQ ID NO: 37) (SEQ ID NO: 44) VEGF_CKP9.72 RLLEPSWG QWYQS  10.6 10.0 (SEQ ID NO: 260) (SEQ ID NO: 264) VEGF_CKP9.76 HLLLPLWG RWYHS 1 1.3 15.5 (SEQ ID NO: 261) (SEQ ID NO: 43) VEGF_CKP9.96 KLFEPLWG RWYES 1 1.2 18.4 (SEQ ID NO: 39) (SEQ ID NO: 567) *S/N = signal to noiseratio as compared to BSA control

These clones were selected for further validation in a phage titrationassay, and soluble folded forms corresponding to ten of these sequenceswere generated for further in vitro assessment (see Table 25 below).From this set of variants, VEGF_CKP9.2, VEGF_CKP9.54 and VEGF_CKP9.63exhibited improved potency against VEGF-A compared to parent VEGF-CKP9in in vitro and cellular assays, with IC₅₀ in 100-200 nM range (seeTable 25).

TABLE 25 Inhibitory activity in phage competition ELISA and VEGF-A-KDRinteraction ELISA phage ELISA Cellular VARIANT IC₅₀ (μM) IC₅₀ (nM)VEGF_CKP9 1.36 11700 VEGF_CKP9.2 0.168  270 VEGF_CKP9.3 1.50 N/DVEGF_CKP9.4 1.95 N/D VEGF_CKP9.20 1.34 N/D VEGF_CKP9.22 >100 N/DVEGF_CKP9.54 0.45  188 VEGF_CKP9.59 5.82 N/D VEGF_CKP9.63 0.146  140VEGF_CKP9.96 49.00 N/D

To enhance potency, we selected the lead 9.54 and 9.63 molecules andgenerated new phage libraries based on these frameworks in which loop 2was randomized. The new libraries were panned against hVEGF-A andyielded a number of loop 2 variants which demonstrated significantlyimproved potency against VEGF-A compared to parent 9.54 and 9.63molecules (see Tables 26 and 27 below, respectively), with the mostpotent molecules exhibiting IC₅₀ in 0.5-2 nM range (see Table 28 below).

TABLE 26Affinity-matured VEGF-A binding loop 2 variants based on 9.54 frameworkVARIANT LOOP 1 LOOP 2 LOOP 5 n ELISA S/N* VEGF_CKP9 QLMQPFWG KQDSD HWYQS(SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 10) 93) 17) VEGF_CKP9.54 NIMLPFWGKQDSD QYYQS (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 33) 93) 45)VEGF_CKP9.54.1 NIMLPFWG GQSFE QYYQS 80 2.4 29.9 (SEQ ID NO: (SEQ ID NO:(SEQ ID NO: 33) 94) 45) VEGF_CKP9.54.2 NIMLPFWG GLDYD QYYQS  1 0.1 26  (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 33) 95) 45) VEGF_CKP9.54.12 NIMLPFWGGPELN QYYQS  1 2.4 38.8 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 33) 298) 45)VEGF_CKP9.54.14 NIMLPFWG QADYA QYYQS  1 2.5 23.5 (SEQ ID NO: (SEQ ID NO:(SEQ ID NO: 33) 299) 45) VEGF_CKP9.54.16 NIMLPFWG GVDYL QYYQS  1 2.430.8 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 33) 300) 45) VEGF_CKP9.54.31NIMLPFWG GTNFL QYYQS  1 2.3 32.5 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 33)301) 45) VEGF_CKP9.54.44 NIMLPFWG SRDFD QYYQS  1 2.4 34.4 (SEQ ID NO:(SEQ ID NO: (SEQ ID NO: 33) 302) 45) VEGF_CKP9.54.48 NIMLPFWG NRDFLQYYQS  1 2.5 34.9 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 33) 303) 45)VEGF_CKP9.54.51 NIMLPFWG GWDQF QYYQS  1 2.5 44.5 (SEQ ID NO: (SEQ ID NO:(SEQ ID NO: 33) 304) 45) VEGF_CKP9.54.56 NIMLPFWG GKDFH QYYQS  1 2.335.8 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 33) 305) 45) VEGF_CKP9.54.59NIMLPFWG GPDLQ QYYQS  1 2.3 35.4 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 33)96) 45) VEGF_CKP9.54.64 NIMLPFWG SGDFA QYYQS  1 2.2 22.4 (SEQ ID NO:(SEQ ID NO: (SEQ ID NO: 33) 306) 45) VEGF_CKP9.54.69 NIMLPFWG GKELNQYYQS  1 2.5 21.7 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 33) 307) 45)VEGF_CKP9.54.76 NIMLPFWG GWSMD QYYQS  1 2.7 42.2 (SEQ ID NO: (SEQ ID NO:(SEQ ID NO: 33) 308) 45) VEGF_CKP9.54.87 NIMLPFWG GYDLQ QYYQS  1 2.426.1 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 33) 309) 45) VEGF_CKP9.54.90NIMLPFWG GRDFE QYYQS  1 2.3 29.5 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 33)97) 45) *S/N = signal to noise ratio as compared to BSA control

TABLE 27 Affinity-matured VEGF-A binding loop 2 variants based onVEGF_CKP9.63 framework *S/N =signal to noise ratio as compared to BSA control VARIANT LOOP 1 LOOP 2Loop5 ELISA S/N* VEGF_CKP9 QLMQPFWG GRDLQ HWYQS (SEQ ID NO: (SEQ ID NO:(SEQ ID NO: 10) 322) 17) VEGF_CKP9.63 DVMQPYWG GVDLS HWYNS (SEQ ID NO:(SEQ ID NO: (SEQ ID NO: 35) 323) 46) VEGF_CKP9.63.1 DVMQPYWG GPDID HWYNS2.0 25.2 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 35) 118) 46) VEGF_CKP9.63.2DVMQPYWG GDDLE HWYNS 2.0 15.0 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 35)324) 46) VEGF_CKP9.63.3 DVMQPYWG GVDMT HWYNS 1.7 20.7 (SEQ ID NO:(SEQ ID NO: (SEQ ID NO: 35) 325) 46) VEGF_CKP9.63.12 DVMQPYWG GMDIEHWYNS 2.6 39.9 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 35) 326) 46)VEGF_CKP9.63.14 DVMQPYWG DGDYQ HWYNS 1.5 21.3 (SEQ ID NO: (SEQ ID NO:(SEQ ID NO: 35) 327) 46) VEGF_CKP9.63.15 DVMQPYWG GNDVS HWYNS 1.5 21.4(SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 35) 328) 46) VEGF_CKP9.63.16DVMQPYWG GRDMD HWYNS 2.2 10.3 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 35)119) 46) VEGF_CKP9.63.18 DVMQPYWG AGDEL HWYNS 2.3 17.3 (SEQ ID NO:(SEQ ID NO: (SEQ ID NO: 35) 329) 46) VEGF_CKP9.63.24 DVMQPYWG GLDEEHWYNS 1.6 20.4 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 35) 330) 46)VEGF_CKP9.63.27 DVMQPYWG DGDFD HWYNS 2.1 26.0 (SEQ ID NO: (SEQ ID NO:(SEQ ID NO: 35) 331) 46) VEGF_CKP9.63.30 DVMQPYWG AGDFE HWYNS 2.0 26.0(SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 35) 332) 46) VEGF_CKP9.63.37DVMQPYWG EMDFD HWYNS 0.6  8.6 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 35)120) 46) VEGF_CKP9.63.39 DVMQPYWG GNSFE HWYNS 1.6 18.9 (SEQ ID NO:(SEQ ID NO: (SEQ ID NO: 35) 333) 46) VEGF_CKP9.63.42 DVMQPYWG GQDLTHWYNS 1.7 23.1 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 35) 334) 46)VEGF_CKP9.63.44 DVMQPYWG GENLA HWYNS 1.7 19.5 (SEQ ID NO: (SEQ ID NO:(SEQ ID NO: 35) 335 46) VEGF_CKP9.63.47 DVMQPYWG GQDYN HWYNS 1.7 20.7(SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 35) 336) 46) VEGF_CKP9.63.50DVMQPYWG GADLS HWYNS 0.9 12.7 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 35)337) 46) VEGF_CKP9.63.54 DVMQPYWG GFDMD HWYNS 1.4 19.9 (SEQ ID NO:(SEQ ID NO: (SEQ ID NO: 35) 338) 46) VEGF_CKP9.63.56 DVMQPYWG GESLSHWYNS 1.8  8.4 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 35) 211) 46)VEGF_CKP9.63.62 DVMQPYWG DLNYE HWYNS 1.8 25.4 (SEQ ID NO: (SEQ ID NO:(SEQ ID NO: 35) 339) 46) VEGF_CKP9.63.65 DVMQPYWG GRDLQ HWYNS 2.0 27.1(SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 35) 322) 46) VEGF_CKP9.63.69DVMQPYWG GVDLS HWYNS 2.9 23.7 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 35)323) 46) VEGF_CKP9.63.87 DVMQPYWG GPDID HWYNS 0.9  8.6 (SEQ ID NO:(SEQ ID NO: (SEQ ID NO: 35) 118) 46)

TABLE 28Inhibitory activity of VEGF_CKP9.54- and VEGF_CKP9.63-derived loop 2 variantsagainst VEGF-A KDR-VEGF Cellular IC₅₀ IC₅₀ VARIANT LOOP 1 LOOP 2 LOOP 5(nm) (nm) VEGF_CKP9 QLMQPFWG KQDSD HWYQS 569 11700 (SEQ ID NO:(SEQ ID NO: (SEQ ID NO: 10) 93) 17) VEGF_CKP9.54 NIMLPFWG KQDSD QYYQS   5.8 188 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 33) 93) 45)VEGF_CKP9.54.1 NIMLPFWG GQSFE QYYQS    0.2 1.47 (SEQ ID NO: (SEQ ID NO:(SEQ ID NO: 33) 94) 45) VEGF_CKP9.54.2 NIMLPFWG GLDYD QYYQS    0.2 4.3(SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 33) 95) 45) VEGF_CKP9.54.59 NIMLPFWGGPDLQ QYYQS    0.5 3.06 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 33) 96) 45)VEGF_CKP9.54.90 NIMLPFWG GRDFE QYYQS    0.2 1.35 (SEQ ID NO: (SEQ ID NO:(SEQ ID NO: 33) 97) 45) VEGF_CKP9.63 DVMQPYWG KQDSD HWYNS   10.8 140(SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 35) 93) 46) VEGF_CKP9.63.1 DVMQPYWGGENFL HWYNS    0.4 0.49 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 35) 117) 46)VEGF_CKP9.63.27 DVMQPYWG GRDMD HWYNS    0.3 5.28 (SEQ ID NO: (SEQ ID NO:(SEQ ID NO: 35) 119) 46) VEGF_CKP9.63.44 DVMQPYWG EMDFD HWYNS    0.22.05 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 35) 120) 46) VEGF_CKP9.63.69DVMQPYWG GESLS HWYNS    2.1 26.4 (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 35)211) 46) VEGF_CKP9.63.12 DVMQPYWG GPDID HWYNS    0.7 1.83 (SEQ ID NO:(SEQ ID NO: (SEQ ID NO: 35) 118) 46)

The affinities/potencies of VEGF_CKP9.63.1, VEGF_CKP9.63.27,VEGF_CKP9.63.44, VEGF_CKP9.63.69, and VEGF_CKP9.63.12 for hVEGF-A(8-109) are shown below in Table 29.

TABLE 29 VARIANT k_(a) k_(d) K_(D) EM63 0.16 ± 0.03 1.6 ± 0.5   100 ± 9nM L2.9.63.1 6 ± 1 0.37 ± 0.13 5.8 ± 1.2 L2.9.63.12 8 ± 1 0.10 ± 0.041.1 ± 0.2 L2.9.63.27 11 ± 4  0.15 ± 0.04 1.4 ± 0.2 L2.9.63.44 10 ± 2 0.11 ± 0.02 1.2 ± 0.2 L2.9.63.69 3 ± 1 0.20 ± 0.04 6.9 ± 0.7

Variants VEGF_CKP9.54.90 (see row 2 of Table 26) and VEGF_CKP9.63.12(see row 6 of Table 27), as well as parental variants VEGF_CKP9.54 (seerow 12 of Table 24) and VEGF_CKP9.63 (see row 15 of Table 24), bind withsimilar affinity to human, mouse, rat and rabbit VEGF-A, as determinedby surface plasmon resonance. See Table 30 below.

TABLE 30 Binding kinetics and affinities of VEGF_CKP9.54.90,VEGF_CKP9.63.12, VEGF_CKP9.54, and VEGF_CKP9.63 for various VEGFisoforms. KD VARIANT VEGF Isoform ka ka (error) kd kd (error) KD (nM)(error) 9.54 human 8-109 1.26 × 10⁶ 1.10 × 10⁵ 2.18 × 10⁻¹ 1.19 × 10⁻²175.88 18.21 human 165 8.23 × 10⁵ 1.11 × 10⁵ 1.49 × 10⁻¹ 1.29 × 10⁻²189.67 34.57 mouse 164 8.26 × 10⁵ 2.93 × 10⁴ 2.07 × 10⁻¹ 2.44 × 10⁻²249.87 21.19 rat 1.93 × 10⁶ 8.91 × 10⁵ 2.96 × 10⁻¹ 9.36 × 10⁻² 175.6426.27 rabbit 2.10 × 10⁶ 7.12 × 10⁵ 2.74 × 10⁻¹ 9.14 × 10⁻² 133.22 9.479.54.90 human 8-109 5.15 × 10⁷ 1.62 × 10⁷ 4.05 × 10⁻² 8.77 × 10⁻³ 0.870.13 human 165 1.58 × 10⁷ 3.87 × 10⁶ 1.33 × 10⁻² 1.70 × 10⁻³ 0.89 0.10mouse 164 8.71 × 10⁶ 2.79 × 10⁶ 1.01 × 10⁻² 2.12 × 10⁻³ 1.31 0.24 rat1.72 × 10⁷ 7.43 × 10⁶ 1.35 × 10⁻² 3.33 × 10⁻³ 0.90 0.14 rabbit 5.15 ×10⁷ 1.36 × 10⁷ 6.75 × 10⁻² 8.99 × 10⁻³ 1.14 0.18 9.63 human 8-109 6.62 ×10⁵ 9.83 × 10⁴ 1.81 × 10⁻¹ 2.56 × 10⁻² 281.44 43.53 human 165 3.40 × 10⁵2.79 × 10⁴ 1.30 × 10⁻¹ 1.33 × 10⁻² 381.89 19.15 mouse 164 5.57 × 10⁵4.60 × 10⁴ 1.60 × 10⁻¹ 1.35 × 10⁻² 288.75 11.08 rat 4.56 × 10⁵ 1.49 ×10⁵ 2.46 × 10⁻¹ 9.23 × 10⁻² 523.93 25.06 rabbit 4.24 × 10⁵ 3.22 × 10⁴1.30 × 10⁻¹ 1.95 × 10⁻² 311.43 52.25 9.63.12 human 8-109 6.54 × 10⁶ 7.25× 10⁵ 2.50 × 10⁻² 3.45 × 10⁻³ 3.20 0.22 human 165 4.65 × 10⁶ 8.39 × 10⁵2.01 × 10⁻² 3.95 × 10⁻³ 4.32 0.15 mouse 164 1.04 × 10⁶ 2.81 × 10⁵ 1.32 ×10⁻² 3.04 × 10⁻³ 13.07 1.74 rat 6.44 × 10⁶ 3.87 × 10⁶ 2.59 × 10⁻² 1.01 ×10⁻² 5.74 1.54 rabbit 6.91 × 10⁶ 1.35 × 10⁶ 1.78 × 10⁻² 4.46 × 10⁻³ 2.540.40

VEGF_CKP9.54.90 is also highly selective to VEGF-A and does not bind toor inhibit the activity of other VEGF isoforms such as VEGF-B, VEGF-Cand VEGF-D or other growth factors such as P1GF, EGF, NGF, IGF and PDGF.As shown in FIGS. 2A and 2B, the variant VEGF_CKP9.54.90 disrupts theinteraction between VEGF-A and KDR as well as the interaction betweenVEGF-A and Flt-1, but not disrupt the interaction between VEGF-B andFlt-1, between VEGF-C and Flt-4, between VEGF-D and Flt-4, betweenPIGF-2 and Flt-1, between EGF and EGFR, between PDGF and PDGFR, betweenNGF and NGFR, or between IGF and IGFR.

Unlike EETI-II, VEGF_CKP9.54.90, VEGF_CKP9.54, and VEGF_CKP9.63.12 donot inhibit trypsin protease activity as measured in a peptide substratecleavage assay (Stanger et al. (2014) FEBS Lett. 588 (23), 4487-96). SeeFIGS. 3A and 3B. However, VEGF_CKP9.54.90 and VEGF_CKP9.63.12 maintain adegree of resistance to trypsin digestion (see FIGS. 4A, 4B, and 4C).Approximately 20% of VEGF_CKP9.54.90 was cleaved at Arg13 within loop 2after 24 h incubation with trypsin at 37° C.

VEGF_CKP9.54, VEGF_CKP9.63, and VEGF_CKP9.54.90 each contains roughly a3-turn alpha-helix and each adopts a disulfide signature that isdistinct from that of wild-type EETI-II (C1-C4, C2-C3, C5-C6 forVEGF_CKP9.54.90 vs. C1-C4, C2-05, C3-C6 for wild-type EETI-II). SeeFIGS. 5A and 5B. On one side of the helix, VEGF_CKP9.54.90 forms a fusedbicyclic structure that is bridged by two disulfide bonds (C1-C4 andC2-C3), encompassing loops 1, 2 and 3, and ˜1.5 turn of the alpha-helix.Loop 5 forms on the opposite side of the helix and is constrained byC5-C6 disulfide bond.

The co-crystal structures of VEGF_CKP9.54, VEGF_CKP9.63, andVEGF_CKP9.54.90 in complex with VEGF-A were obtained. co-crystalstructures of VEGF_CKP9.54, VEGF_CKP9.63, and VEGF_CKP9.54.90 in complexwith VEGF-A are highly similar. See FIGS. 5A, 5B, 6A and 6B for theco-crystal structure of VEGF_CKP9.54 in complex with VEGF-A. Given thatthe structures of VEGF_CKP9.54, VEGF_CKP9.63, and VEGF_CKP9.54.90 arehighly similar, (see FIGS. 5A, 5B, 6A and 6B) further studies wereperformed with VEGF_CKP9.54.90. The helix defined by residuesPhe15-Tyr26 of VEGF_CKP9.54.90 forms extensive hydrophobic and polarinteractions with the VEGF-A surface (see Table 31 below). Additionally,there is a network of backbone H-bonds which forms within and stabilizesthe ˜3-turn alpha-helix. In general, VEGF_CKP9.54.90 exhibits a compactand rigid structure, stabilized by intramolecular polar and hydrophobiccontacts, including backbone-backbone, side chain-backbone and sidechain-side chain interactions (Table 32). The surface of VEGF_CKP9.54.90that contacts VEGF-A is mainly hydrophobic in nature with few polar sidechains (Table 31), whereas the opposite surface of the peptide that isnot interacting with VEGF-A is solvent-exposed and primarily polar innature.

TABLE 31 VEGF_CKP9.54.90 residues that are within 4 Å of the VEGF-Adimer VEGF_CKF9.54.90 residues within 4 Å of VEGFA VEGF_CKP9.54.90VEGF_CKP9.54.90 (Chain 1) (Chain 2) I4 I4 M5 M5 L6 L6 P7 P7 F8 F8 W9 W9R13 R13 D14 D14 F15 F15 L18 L18 A19 A19 V22 V22 C23 C23 Y25 Y25 Y26 Y26Q27 Q27 S28 S28 G30 G30

TABLE 32 Summary of VEGF_CKP9.54.90 intra-molecular interactions Residue1 Residue 2 Comments Cys2 Cys21 Disulfide Asn3 Trp9 Main chain H-bondAsn3 Met5 Asp3 makes H-bond with M5 main chain nitrogen Asn3 Leu6 Mainchain H-bond Leu6 Trp9 Main chain H-bond Pro7 Gly10 Main chain H-bondPhe8 Cys11 Main chain H-bond Phe8 Leu18 Van der Waals interaction Trp9Ile4, Leu18, Core Trp makes a network of Val22, Tyr25, Van der Waalsinteractions Tyr26 Cys11 Cys17 Disulfide Gly12 Asp14 Main chain H-bondAsp14 Glu16, C17 Asp14 makes stabilizing H-bond with N-terminus of helixPhe15 Leu18, Ala19 Van der Waals interactions stabilizing helix Leu18Val22 Van der Waals interactions stabilizing helix Val22 Tyr26 Van derWaals interactions stabilizing helix Phe15-Tyr26 Network of backboneH-bonds form stabilizing a ~3-turn helix Cys23 Cys29 Disulfide Tyr25Tyr26 Van der Waals interactions stabilizing helix Gln27 Cys23 Mainchain H-bond Ser28 Cys23 Main chain H-bond

The binding interface of VEGF_CKP9.54.90 on VEGF-A overlaps with that ofthe natural receptors and G6.31 antibody (FIGS. 7A-7F and 8A-C). Contactresidues on VEGF-A that are in the peptide interface are summarized inTable 33 and shown in FIG. 9. The binding epitope of VEGF_CKP9.54.90 onVEGF-A is distinct from that of ranibizumab and bevacizumab (FIGS. 10A,10B, and 10C), which do not bind to mouse or rat VEGF-A because theirinteraction with human VEGF-A is dependent on a key Gly88 residue thatis substituted with Ser in rodents. The binding mode of VEGF_CKP9.54.90suggests that it is not substantially dependent on Gly88, and thisnotion is validated by the observation that the peptide boundefficiently to both human and rodent VEGF-A. Site-directed mutagenesiswas utilized to validate a number of contacts in the protein—peptideinterface observed from the crystal structure. As expected, Y21A, Q89Aand F17A/M81A mutations on VEGF-A led to reduced binding ofVEGF_CKP9.54.90 on VEGF-A. See FIGS. 11A and 11B. However, K48A mutationenhanced the binding of VEGF_CKP9.54.90 by ˜2-3 fold, a behavior that issimilar to that observed with the G6.31 antibody (Fuh et al. (2006) J.Biol. Chem. 281, 6625-6631). See Table 34 below and FIGS. 11A and 11B.

TABLE 33 VEGF-A dimer residues that are within 4 Å of VEGF_CKP9.54.90VEGF-A residues within 4 Å of VEGF_CKP9.54.90 VEGF-A VEGF-A (Dimer ChainA) (Dimer Chain B) V14 V15 F17 F17 M18 M18 D19 Y21 Y21 Q22 Q22 Y25 Y25I46 I46 K48 K48 N62 N62 D63 D63 L66 L66 M81 M81 I83 I83 K84 K84 P85 P85H86 H86 Q87 Q87 G88 G88 Q89 Q89 I91 C104 R105 P106 P106

TABLE 34 Binding kinetics and affinities of VEGF_CKP9.54.90,VEGF_CKP9.63.12, VEGF_CKP9.54, and VEGF_CKP9.63 for various hVEGF-Amutants. VEGF ka kd KD KD VARIANT MUTANT ka (error) kd (error) (nM)(error) 9.54.90 WT 7.81 × 10⁷ 1.86 × 10⁷ 0.0300 9.79 × 10⁻³ 0.37 0.09Y21A 3.72 × 10⁷ 1.30 × 10⁷ 0.1202 2.44 × 10⁻² 3.50 0.30 K48A 6.04 × 10⁷2.12 × 10⁷ 0.0116 3.77 × 10⁻³ 0.19 0.02 Q89A 1.71 × 10⁷ 8.38 × 10⁶0.1458 5.99 × 10⁻² 8.80 0.45 F17A/M81A 9.63.12 WT 8.43 × 10⁶ 1.47 × 10⁶0.0096 0.0035 1.07 0.20 Y21A 1.99 × 10⁷ 6.84 × 10⁶ 1.48 × 10⁻¹ 3.13 ×10⁻² 9.84 4.41 K48A 5.30 × 10⁷ 3.77 × 10⁷ 0.017 0.013  0.33 0.01 Q89A7.43 × 10⁶ 2.09 × 10⁶ 0.47 2.71 × 10⁻¹ 36.49 1.62 F17A/M81A 9.54 WT 4.10× 10⁶ 8.59 × 10⁵ 0.2349 0.0778 55 7 Y21A 6.63 × 10⁵ 6.39 × 10³ 0.21010.0389 317 56 K48A 2.64 × 10⁶ 2.24 × 10⁵ 0.0587 0.0010 22 3 Q89A 3.37 ×10⁵ 1.41 × 10⁵ 0.7932 0.5101 1882 460 F17A/M81A 9.63 WT 1.57 × 10⁶ 3.14× 10⁵ 0.1624 0.05  100.34 9.40 Y21A 7.52 × 10⁵ 2.45 × 10⁵ 0.4814 2.13 ×10⁻¹ 584.85 112.171 K48A 5.72 × 10⁵ 1.75 × 10⁵ 0.02 5.24 × 10⁻³ 28.4 4.8Q89A 2.66 × 10⁵ 8.01 × 10⁴ 0.51 1.22 × 10⁻¹ 1999.8 127.5 F17A/M81A

Next, VEGF-A binding variants VEGF_CKP9.54.90, VEGF_CKP9.63.12, andVEGF_CKP9.63.44 were assessed for their in vivo efficacy in a VEGF-Adriven model of choroidal neovascularization. Laser-burnt spots werecreated in rat eyes and the formation of new vessels was monitored aftera 14-day period in the presence and absence of peptide that wasadministered intravitreally at different intervals. PeptideVEGF_CKP9.54.90 demonstrated effective inhibition of laser-inducedchoroidal neovascularization in rat eyes, as measured by the significantreduction observed in neovascular area in peptide-treated eyes comparedto control eyes. See FIG. 12.

The co-crystal structure of VEGF_CKP9.54.90 in complex with VEGF-Arevealed that the native amino acid residues in loops 3 and 4 are notnecessarily in optimal orientations for binding to VEGF-A (see FIG. 9)and could be modified to enhance their interaction with the VEGF-Asurface or to elicit intramolecular interactions within the peptide thatcould improve peptide folding and stability. Therefore, with the goal offurther improving the potency and behavior of the lead molecules, newphage libraries were constructed based on the sequences of 9.54, 54.1and 9.63, 63.12 in which only loops 3 and 4 were randomized. Thesespecific frameworks, though slightly weaker than the lead molecules,were selected in order to allow for a sufficient dynamic range in theassay to detect improvement in affinity. Many new clones containingvariations in loops 3 and 4 only were identified that showed improvedbinding to VEGF-A. Fourteen of the obtained sequences were selected andgrafted onto loops 3 and 4 within the lead VEGF_CKP9.54.90 orVEGF_CKP9.63.12 molecules, and the corresponding soluble molecules werethen generated in folded form. The amino acid sequences of the fourteenaffinity-matured variants are provided in Table 35 below.

TABLE 35 Affinity-matured VEGF-A binding loop 3/loop 4 variants based onVEGF_CKP9.54.90 or VEGF_CKP9.63.12 frameworks VARIANT LOOP 1 LOOP 2LOOP 3 LOOP 4 LOOP 5 9.54.90.7 NIMLPFWG GRDFE LQQ I QYYQS(SEQ ID NO: 33) (SEQ ID NO: 97) (SEQ ID NO: 45) 9.54.90.10 NIMLPFWGGRDFE VER I QYYQS (SEQ ID NO: 33) (SEQ ID NO: 97) (SEQ ID NO: 45)9.54.90.12 NIMLPFWG GRDFE MSD I QYYQS (SEQ ID NO: 33) (SEQ ID NO: 97)(SEQ ID NO: 45) 9.54.90.13 NIMLPFWG GRDFE MNQ I QYYQS (SEQ ID NO: 33)(SEQ ID NO: 97) (SEQ ID NO: 45) 9.54.90.25 NIMLPFWG GRDFE MQT I QYYQS(SEQ ID NO: 33) (SEQ ID NO: 97) (SEQ ID NO: 45) 9.54.90.31 NIMLPFWGGRDFE VYQ I QYYQS (SEQ ID NO: 33) (SEQ ID NO: 97) (SEQ ID NO: 45)9.54.90.44 NIMLPFWG GRDFE FIN I QYYQS (SEQ ID NO: 33) (SEQ ID NO: 97)(SEQ ID NO: 45) 9.54.90.53 NIMLPFWG GRDFE  VSQ I QYYQS (SEQ ID NO: 33)(SEQ ID NO: 97) (SEQ ID NO: 45) 9.54.90.55 NIMLPFWG GRDFE VTE I QYYQS(SEQ ID NO: 33) (SEQ ID NO: 97) (SEQ ID NO: 45) 9.54.90.62 NIMLPFWGGRDFE FYE I QYYQS (SEQ ID NO: 33) (SEQ ID NO: 97) (SEQ ID NO: 45)9.54.90.67 NIMLPFWG GRDFE MEQ I QYYQS  (SEQ ID NO: 33) (SEQ ID NO: 97)(SEQ ID NO: 45) 9.54.90.71 NIMLPFWG GRDFE VYR I QYYQS  (SEQ ID NO: 33)(SEQ ID NO: 97) (SEQ ID NO: 45) 9.63.12.8 DVMQPYWG GPDID FVR L HWYNS(SEQ ID NO: 35) (SEQ ID NO: 118) (SEQ ID NO: 46) 9.63.12.12 DVMQPYWGGPDID LSN I HWYNS (SEQ ID NO: 35) (SEQ ID NO: 118) (SEQ ID NO: 46)

All soluble molecules containing L3/L4 variations showed improvedpotency in the cellular assay relative to 54.90 or 63.12.12. Three leadmolecules, VEGF_CKP9.54.90.67, VEGF_CKP9.54.90.53 and VEGF_CKP9.63.12.12had cellular IC₅₀ values in the range of about 0.5 to about 1 nM. SeeFIG. 13 and Table 36 below.

TABLE 36 IC₅₀ values for variants in Table 35 FOLD IMPROVEMENT RELATIVETO Cellular VARIANT VARIANT IC₅₀ (nM) 9.54.90 9.54.90 1.35 1 9.54.90.121.20 1.125 9.54.90.13 0.96 1.41 9.54.90.25 1.26 1.07 9.54.90.44 1.161.16 9.54.90.62 0.92 1.47 9.54.90.67 1.10 1.23 9.63.12 1.83 0.749.63.12.8 N/D 9.63.12.12 0.56 2.41

The co-crystal structure of VEGF_CKP9.63 in complex with VEGF-A revealedthat Tyr residue at position 8 within loopl could form a hydrogen bondwith the side chain of Gln22 on VEGF-A. See FIGS. 14A, 14B, 14C and 14D.In variants derived from VEGF_CKP9.54 (such as the variants in Table 35)the amino acid at position 8 is Phe. Therefore, we sought to mutate Phe8to Tyr I in some of the variants in Table 35, with the goal of improvingaffinity and/or solubility of the resulting F8Y variant. The F8Ymutation showed a modest improvement on affinity/potency of some of themolecules (e.g., VEGF_CKP9.54.1.F8Y, VEGF_CKP9.54.90.F8Y, andVEGF_CKP9.54.90.67.F8Y), whereas in few other cases it demonstratedminimal or a slightly negative effect (e.g., VEGF_CKP9.54.90.13.F8Y andVEGF_CKP9.54.90.62.F8Y). See Table 37, in which the binding affinitiesof certain variants (as determined by surface plasmon resonance) arecompared, and Table 38 in which the potencies of certain variants (asdetermined by cellular IC₅₀) are compared. The F8Y substitution helpedto improve the solubility of VEGF_CKP9.54.90.67.F8Y by about 2 mg/ml.VEGF_CKP9.54.90.67.F8Y was selected for further follow-up studies.

TABLE 37 Binding kinetics and affinities of VEGF_CKP9.54.1.F8Y,VEGF_CKP9.54.90.F8Y, VEGF_CKP9.54.90.67.F8Y, VEGF_CKP9.54.90.13.F8Y andVEGF_CKP9.54.90.62.F8Y for VEGF-A VARIANT k_(a) k_(d) K_(D) VEGF_CKP91.2 ± 0.3 40 ± 20 5 ± 1 μM VEGF_CKP9.54 3.4 ± 0.2 2.3 ± 0.8 44 ± 6 nM VEGF_CKP9.54.1 15 ± 2  0.36 ± 0.17 2.2 ± 0.7 nM VEGF_CKP9.54.1.F8Y 53 ±12 0.38 ± 0.06 0.78 ± 0.11 nM VEGF_CKP9.54.90 63 ± 16 0.17 ± 0.05 0.40 ±0.08 nM VEGF_CKP9.54.90.F8Y 70 ± 10 0.27 ± 0.01 0.40 ± 0.05 nMVEGF_CKP9.54.90-Alkyn  50 ± 0.3  0.27 ± 0.004 0.49 ± 0.05 nM

The oxidative stability of various CKP variants was assayed as follows:5 μl of 11 mM AAPH (Calbiochem catalog no. 100110) in water was added to50 uL of variant peptide sample (prepared as 1 mg/mL peptide in 20 mMhistidine acetate pH 5.5) and the mix was incubated for 16 hours at 40°C. At the end of the incubation, the sample was quenched by addition of27.5 uL of 40 mM methionine, followed by addition of 160 ul of 20 mMHistidine acetate, 100 mM sucrose at pH 5.5 to dilute the samples. Thereactions were analyzed by LC-MS.

It was observed that VEGF_CKP9.54.90 underwent ˜30% oxidation at Met5within loop 1. Replacement of Met 5 with the unnatural amino acidnorleucine rendered VEGF_CKP9.54.90 completely resistant to oxidation.The replacement of Met5 with norleucine also had a favorable effect onbinding efficiency (˜2-fold improvement). Variants VEGF_CKP9.54.90.67F8Y M5Nle, VEGF_CKP9.54.90.53 M5Nle and VEGF_CKP9.63.12.12 M5Nle wereproduced. All three Met5Nle of the Met5Nle variants showed modestimprovement in cellular potency by ˜1.5-2× compared to their parentmolecules.

Next, the effect of naphthalene-based amino acid substitutions at Trp9in loop 1 of VEGF_CKP9.54.90 on VEGF-A binding affinity was assessed.The crystal structures of VEGF_CKP9.54.90 complexed with VEGF indicatethat the Trp9 residue of VEGF_CKP9.54.90 (and variants derivedtherefrom) interacts with the VEGF-A surface, with residual space thatmight allow larger ring systems to fit in. To test this hypothesis, wegenerated soluble variants of VEGF_CKP9.54.90 in which the indole ringof Trp9 was replaced with 1- or 2-naphthyl isomers. These moleculesshowed reduced cellular potency relative to parent VEGF_CKP9.54.90.Further data regarding the potency of VEGF_CKP9.54.90-derived variantscomprising the F8Y substitution and/or an unnatural amino acidsubstitution are provided in Table 38:

TABLE 38 IC₅₀ values for VEGF_CKP9.54.90-derived variants comprising theF8Y substitution and/or an unnatural amino acid substitution. FOLDIMPROVEMENT RELATIVE TO Cellular VARIANT VARIANT IC₅₀ (nM) 9.54.909.54.90 1.35 1 9.54.90.F8Y 1.58 0.85 9.54.90.F7Y.Δ2G 3.01 0.449.54.90.M5.Nle 1.95 0.69 9.54.90.Naph1 5.42 0.24 9.54.90.Naph2 17.1 0.089.54.90.13 0.96 1.41 9.54.90.13.F8Y 2.02 0.67 9.54.90.62 0.92 1.479.54.90.62.F8Y 1.14 1.18 9.54.90.67 1.1 1.23 9.54.90.67.F8Y 0.66 2.05

Shortening the lead VEGF_CKP9.54.90. F8Y by trimming the two glycineresidues at the N- and C-termini to generate variant VEGF_CKP9.54.90 F7YΔ2G) resulted in a slight reduction of cellular potency relative toVEGF_CKP9.54.90. See Table 38 above.

Example 2B: Generation of VEGF-A-Binding Non-Naturally Occurring EETI-IIVariants Comprising C-Terminal Amino Acid Extensions

To identify additional peptide variants with enhanced affinity forVEGF-A, we selected the 9.54 (SEQ ID NO: 52), 9.54.1 (SEQ ID NO: 99)molecules and generated new phage libraries based on these frameworks inwhich two additional amino acids were added to their C-termini.

From the 9.54 library, twenty-two clones whose binding signals forhVEGF-A (8-109) were more than 3 times higher than to BSA (background)were identified (Table 39). These hits contained variations in aminoacid composition within loop 2, within loops 2 and 4, or within loops 2,4, and 5.

TABLE 39 C-terminal Two-residue Extension Variants Based on 9.54 SEQ IDCLONE ID AMINO ACID SEQUENCE NO 9.54 GCNIMLPFWGCKQDSDCLAGCVCQYYQSCG  529.54-28 GCNIMLPFWGCKQDFDCLAGCICQYYQSCGFH 455 9.54-39GCNIMLPFWGCKQDFDCLAGCICQYYQSCGGE 457 9.54-10GCNIMLPFWGCKQDSDCLVGCICQYYQSCGSI 458 9.54-32GCNIMLPFWGCKQDFDCLAGCVCQYYQSCGGR 459 9.54-13GCNIMLPFWGCKQDFDCLAGCVCQYYQSCGRP 460 9.54-6GCNIMLPFWGCKQDFDCLAGCVCQYYQSCGQY 461 9.54-24GCNIMLPFWGCKQDSDCLAGCVCQYYQSCGEN 462 9.54-34GCNIMLPFWGCKQDFDCLAGCVCQYYQSCGDT 463 9.54-9GCNIMLPFWGCKQDFDCLAGCVCQYYQSCGQH 464 9.54-12GCNIMLPFWGCKQDSDCLAGCICQYYQSCGQN 465 9.54-17GCNIMLPFWGCKQDSDCLAGCVCQYYQSCGEE 466 9.54-19GCNIMLPFWGCKQDSDCLAGCVCQYYQSCGDD 467 9.54-43GCNIMLPFWGCKQDSDCLAGCVCQYYQSCGDG 468 9.54-5GCNIMLPFWGCKQDFDCLAGCVCQYYQSCGLE 469 9.54-1GCNIMLPFWGCKQDSDCLAGCVCQYYQSCGTD 470 9.54-4GCNIMLPFWGCKQDSDCLAGCVCQYYQSCGSE 471 9.54-15GCNIMLPFWGCKQDSDCLAGCVCQYYQSCGPE 472 9.54-42GCNIMLPFWGCKQDSDCLAGCVCQYYQSCGTN 473 9.54-27GCNIMLPFWGCKQDSDCLAGCVCQYYQSCGPH 474 9.54-2GCNIMLPFWGCKQDSDCLAGCVCQYYQSCGMD 475 9.54-21GCNIMLPFWGCKQDSDCLAGCVCQYYQSCGSD 476

From the 9.54.1 library, clones whose binding signals for hVEGF-A(8-109) were more than 3 times higher than to BSA (background) wereidentified (Table 40).

TABLE 40 C-terminal Two-residue Extension Variants Based on 9.54.1 SEQID CLONE ID AMINO ACID SEQUENCE NO 9.54.1 GCNIMLPFWGCGQSFECLAGCVCQYYQSCG 99 9.54.1-2 GCNIMLPFWGCGQSFECLAGCICQYYQSCGIA 477 9.54.1-63GCNIMLPFWGCGQSFECLAGCICQYYQSCGGS 478 9.54.1-36GCNIMLPFWGCGQSFECLAGCICQYYQSCGTR 479 9.54.1-42GCNIMLPFWGCGQSFECLAGCICQYYQSCGLS 533 9.54.1-90GCNIMLPFWGCGQSFECLAGCICQYYQSCGVH 480

Clone 9.54-28 (in Table 39) showed approximately 10-fold improvedbinding affinity for hVEGF-A (8-109) compared to 9.54, as determined byphage competition ELISA (described above). (See FIG. 15). Clones9.54.1-2, 9.54.1-36, 9.54.1-42, 9.54.1-63, and 9.54.1-90 (in Table 40)also showed approximately 10-fold improved binding affinity for hVEGF-A(8-109) compared to 9.54.1, as determined by phage competition ELISA.(See FIG. 15).

Peptides 9.63 (SEQ ID NO: 55), and 9.63.44 (SEQ ID NO: 125) wereselected for further modification as described above. New phagelibraries based on these frameworks were generated in which twoadditional amino acids were added to their C-termini.

From the 9.63 library, 28 clones whose binding signals for hVEGF-A(8-109) were more than 3 times higher than to BSA (background) wereidentified (Table 41). These hits contained variations in amino acidcomposition within loops 2 and 4.

TABLE 41 C-terminal Two-residue Extension Variants Based on 9.63 SEQ IDCLONE ID AMINO ACID SEQUENCE NO 9.63 GCDVMQPYWGCKQDSDCLAGCVCHWYNSCG 559.63-1 GCDVMQPYWGCKQDFDCLAGCVCHWYNSCGPS 481 9.63-4GCDVMQPYWGCEMDFDCLAGCICHWYNSCGFS 482 9.63-7GCDVMQPYWGCEMDFDCLAGCVCHWYNSCGGK 483 9.63-10GCDVMQPYWGCKQDFDCLAGCICHWYNSCGYL 484 9.63-16GCDVMQPYWGCEMDFDCLAGCVCHWYNSCGDL 485 9.63-17GCDVMQPYWGCEMDFDCLAGCVCHWYNSCGEK 486 9.63-19GCDVMQPYWGCKQDSDCLAGCICHWYNSCGTD 487 9.63-20GCDVMQPYWGCEMDFDCLAGCICHWYNSCGQV 488 9.63-21GCDVMQPYWGCEMDFDCLAGCVCHWYNSCGRL 489 9.63-22GCDVMQPYWGCEMDFDCLAGCICHWYNSCGYA 490 9.63-23GCDVMQPYWGCEMDFDCLAGCICHWYNSCGAS 491 9.63-25GCDVMQPYWGCEMDFDCLAGCICHWYNSCGSR 492 9.63-30GCDVMQPYWGCEMDFDCLAGCICHWYNSCGPT 493 9.63-36GCDVMQPYWGCEMDFDCLAGCVCHWYNSCGSL 456 9.63-40GCDVMQPYWGCEMDFDCLAGCVCHWYNSCGWD 494 9.63-45GCDVMQPYWGCEMDFDCLAGCVCHWYNSCGSM 495 9.63-61GCDVMQPYWGCEMDFDCLAGCICHWYNSCGTR 496 9.63-62GCDVMQPYWGCKQDSDCLAGCVCHWYNSCGEN 497 9.63-65GCDVMQPYWGCEMDFDCLAGCVCHWYNSCGNN 498 9.63-66GCDVMQPYWGCEMDFDCLAGCICHWYNSCGPE 499 9.63-67GCDVMQPYWGCEMDFDCLAGCVCHWYNSCGGI 500 9.63-68GCDVMQPYWGCEMDFDCLAGCVCHWYNSCGVE 501 9.63-70GCDVMQPYWGCEMDFDCLAGCVCHWYNSCGPL 503 9.63-72GCDVMQPYWGCEMDFDCLAGCICHWYNSCGTS 527 9.63-74GCDVMQPYWGCEMDFDCLAGCICHWYNSCGRP 504 9.63-77GCDVMQPYWGCEMDFDCLAGCVCHWYNSCGND 505 9.63-79GCDVMQPYWGCEMDFDCLAGCICHWYNSCGLQ 506 9.63-93GCDVMQPYWGCEMDFDCLAGCICHWYNSCGDE 507

From the 9.63.44 library, 17 clones whose binding signals for hVEGF-A(8-109) were more than 3 times higher than to BSA (background) wereidentified (Table 42). These hits contained a variation in amino acidcomposition within loop 4. Clone 9.63.44-55 contained a variation inamino acid composition within loop 2, and clone 9.63.44-10 contained avariation in amino acid composition within loop 3. Interestingly clone9.63.44-12 in Table 42 and clone 9.63-70 in Table 41 have the same aminoacid sequence.

TABLE 42 C-terminal Two-residue Extension Variants Based on 9.63.44 SEQID CLONE ID AMINO ACID SEQUENCE NO 9.63.44GCDVMQPYWGCEMDFDCLAGCVCHWYNSCG 125 9.63.44-2-GCDVMQPYWGCEMDFDCLAGCICHWYNSCGRT 508 A 9.63.44-55GCDVMQPYWGCEIDFDCLAGCVCHWYNSCGQV 509 9.63.44-10-GCDVMQPYWGCEMDFDCLAGCVCHWYNSCGGI 510 A 9.63.44-54GCDVMQPYWGCEMDFDCLAGCVCHWYNSCGYM 511 9.63.44-19GCDVMQPYWGCEMDFDCLAGCVCHWYNSCGGQ 512 9.63.44-44GCDVMQPYWGCEMDFDCLAGCVCHWYNSCGTP 513 9.63.44-14GCDVMQPYWGCEMDFDCLAGCVCHWYNSCGVN 514 9.63.44-73GCDVMQPYWGCEMDFDCLAGCVCHWYNSCGFN 515 9.63.44-16GCDVMQPYWGCEMDFDCLAGCVCHWYNSCGEP 516 9.63.44-80GCDVMQPYWGCEMDFDCLAGCVCHWYNSCGNS 517 9.63.44-41GCDVMQPYWGCEMDFDCLAGCVCHWYNSCGST 518 9.63.44-82GCDVMQPYWGCEMDFDCLAGCVCHWYNSCGRY 519 9.63.44-1GCDVMQPYWGCEMDFDCLAGCICHWYNSCGFS 520 9.63.44-2GCDVMQPYWGCEMDFDCLAGCICHWYNSCGQV 521 9.63.44-3GCDVMQPYWGCEMDFDCLAGCICHWYNSCGYA 522 9.63.44-4GCDVMQPYWGCEMDFDCLAGCICHWYNSCGSR 523 9.63.44-5GCDVMQPYWGCEMDFDCLAGCICHWYNSCGPT 524 9.63.44-6GCDVMQPYWGCEMDFDCLAGCVCHWYNSCGSM 525 9.63.44-7GCDVMQPYWGCEMDFDCLAGCVCHWYNSCGGI 526 9.63.44-8GCDVMQPYWGCEMDFDCLAGCICHWYNSCGTS 527 9.63.44-9GCDVMQPYWGCEMDFDCLAGCICHWYNSCGLQ 528 9.63.44-10GCDVMQPYWGCEMDFDCLVGCVCHWYNSCGDE 529 9.63.44-11GCDVMQPYWGCEMDFDCLAGCVCHWYNSCGDL 530 9.63.44-12GCDVMQPYWGCEMDFDCLAGCVCHWYNSCGPL 503 9.63.44-13GCDVMQPYWGCEMDFDCLAGCVCHWYNSCGQF 531 9.63.44-14GCDVMQPYWGCEMDFDCLAGCICHWYNSCGWK 532

Clones 9.63.44-1 through 9.63.44-14 (in Table 42) showed improvedbinding affinity for hVEGF-A (8-109) compared to 9.63.44, as determinedby phage competition ELISA. (See FIGS. 16A and 16B).

Taken together, the results above indicate that extending lead peptides9.54 (SEQ ID NO: 52), 9.54.1 (SEQ ID NO: 99), 9.63 (SEQ ID NO: 55), and9.63.44 by adding two amino acids to their C-termini produced variantshaving ˜10-fold greater binding affinity for hVEGF-A (8-109).

Next, peptides 9.54.90 (SEQ ID NO: 102) and 63.12.12.M5L (SEQ ID NO:386) were selected for further modification as described above. Brieflynew phage libraries were generated based on 9.54.90 in which twoadditional amino acids, three additional amino acids, or four additionalamino acids were added at the C-terminus. A second set of libraries wasgenerated based on 63.12.12.M5L in which two additional amino acids wereadded at the C-terminus.

From the 9.54.90 libraries comprising 2-amino acid C-terminalextensions, 6 clones whose binding signals for hVEGF-A (8-109) were morethan 3 times higher than to BSA (background) were identified (Table 43).

TABLE 43 C-terminal Two-residue Extension Variants Based on 9.54.90 SEQID CLONE ID AMINO ACID SEQUENCE NO 9.54.90GCNIMLPFWGCGRDFECLAGCVCQYYQSCG 102 9.54.90-GCNIMLPFWGCGRDFECLAGCVCQYYQSCGFH 379 2x28 9.54.90-GCNIMLPFWGCGRDFECLAGCVCQYYQSCGIA 380 2x2 9.54.90-GCNIMLPFWGCGRDFECLAGCVCQYYQSCGGS 381 2x63 9.54.90-GCNIMLPFWGCGRDFECLAGCVCQYYQSCGTR 382 2x36 9.54.90-GCNIMLPFWGCGRDFECLAGCVCQYYQSCGVH 383 2x90 9.54.90-GCNIMLPFWGCGRDFECLAGCVCQYYQSCGLS 384 2x42

From the 9.54.90 libraries comprising 3-amino acid C-terminalextensions, 10 clones whose binding signals for hVEGF-A (8-109) weremore than 3 times higher than to BSA (background) were identified (Table44).

TABLE 44 C-terminal Three-residue Extension Variants Based on 9.54.90SEQ ID CLONE ID AMINO ACID SEQUENCE NO 9.54.90GCNIMLPFWGCGRDFECLAGCVCQYYQSCG 102 9.54.90-GCNIMLPFWGCGRDFECLAGCVCQYYQSCGPLI 369 3X83 9.54.90-GCNIMLPFWGCGRDFECLAGCVCQYYQSCGNYQ 370 3X50 9.54.90-GCNIMLPFWGCGRDFECLAGCVCQYYQSCGPLQ 371 3x49 9.54.90-GCNIMLPFWGCGRDFECLAGCVCQYYQSCGTFQ 372 3x10 9.54.90-GCNIMLPFWGCGRDFECLAGCVCQYYQSCGDLV 373 3x91 9.54.90-GCNIMLPFWGCGRDFECLAGCVCQYYQSCGEHK 374 3x42 9.54.90-GCNIMLPFWGCGRDFECLAGCVCQYYQSCGYLS 375 3x88 9.54.90-GCNIMLPFWGCGRDFECLAGCVCQYYQSCGWDY 376 3x9 9.54.90-GCNIMLPFWGCGRDFECLAGCVCQYYQSCGWPH 377 3x13 9.54.90-GCNIMLPFWGCGRDFECLAGCVCQYYQSCGPHQ 378 3x33

All peptides from the 9.54.90 libraries comprising 4-amino acidC-terminal extensions, contained 3amino acid C-terminal extensions.

From the 63.12.12.M5L libraries comprising 2-amino acid C-terminalextensions, 9 clones whose binding signals for hVEGF-A (8-109) were morethan 3 times higher than to BSA (background) were identified (Table 45).

TABLE 45 C-terminal Two-residue Extension Variants Based on 63.12.12.M5LSEQ ID CLONE ID AMINO ACID SEQUENCE NO 63.12.12.GCDVLQPYWGCGPDIDCLSNCICHWYNSCG 386 M5L 63.12.12.GCDVLQPYWGCGPDIDCLSNCICHWYNSCGRT 387 M5L.2x2 63.12.12.GCDVLQPYWGCGPDIDCLSNCICHWYNSCGWK 388 M5L.2x77 63.12.12.GCDVLQPYWGCGPDIDCLSNCICHWYNSCGPL 389 M5L.2x48 63.12.12.GCDVLQPYWGCGPDIDCLSNCICHWYNSCGDE 390 M5L.2x25 63.12.12.GCDVLQPYWGCGPDIDCLSNCICHWYNSCGQF 391 M5L.2x69 63.12.12.GCDVLQPYWGCGPDIDCLSNCICHWYNSCGEQ 392 M5L.2x12 63.12.12.GCDVLQPYWGCGPDIDCLSNCICHWYNSCGPT 393 M5L.2x30 63.12.12.GCDVLQPYWGCGPDIDCLSNCICHWYNSCGRL 394 M5L.2x21 63.12.12.GCDVLQPYWGCGPDIDCLSNCICHWYNSCGSL 395 M5L.2x29

Example 2C: Characterization of VEGF-A-Binding Non-Naturally OccurringEETI-II Variants Comprising C-Terminal Amino Acid Extensions

The variants provided in Tables 43-45 above are assayed via phagecompetition ELISA as described above to identify variants with greaterbinding affinity for hVEGF-A (8-109).

Clones (e.g., such as those provided in Tables 39-45) demonstratinggreater affinity for hVEGF (8-109), including, e.g., 9.54.1-2,9.54.1-36, 9.54.1-42, 9.54.1-63, and 9.54.1-90, and9.63.44-1-9.63.44-14, are then selected for further in vitroassessments, such as inhibitory activity in phage competition ELISAs andVEGF-KDR interaction ELISAs, as described above.

Clones are then analyzed via surface plasmon resonance to determinetheir affinities for various VEGF isoforms, including hVEGF-A (8-109),hVEGF-A 165, mouse VEGF-A 164, rat VEGF-A, and rabbit VEGF-A.

Further analyses are performed to assess the clones specificity forVEGF-A. For example, competition ELISAs are performed as described abovewith VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGf-2, NGF, EGF, PDGF-β, or IGF-1.

The clones are also assayed for their abilities to inhibit trypsinprotease activity as measured in a peptide substrate cleavage assay(Stanger et al. (2014) FEBS Lett. 588 (23), 4487-96).

Binding kinetics and affinities of the clones for various hVEGF mutants,including, e.g., Y21A, K48A, Q89A, and F17A/M81A, are determined asdescribed above.

Next, the clones are assessed for their in vivo efficacy in a VEGF-Adriven model of choroidal neovascularization, as described above.

The oxidative stability of the variants is assayed as described above.

Example 3: Generation of Non-Naturally Occurring EETI-II Variants thatBind LRP6

The naïve EETI-II libraries described in Example 2A were cycled throughrounds of selection against LRP6 E1E2 protein. Twenty-two unique cloneswere identified which bound LRP6 E1E2 (Table 46). These initial hitscontained variations in amino acid content within loops 1 and 5. Inseveral variants, loop 1 exhibited a longer length compared to that ofthe native EETI-II framework. Notably, the newly evolved sequences thatbound to LRP6 contained a consensus motif in loop 1 (NXI) that issimilar to a motif (NAI) present within the native Dkk1 molecules whichare endogenous LRP6 ligands. The newly evolved variants recapitulated amotif which occurs in natural ligands.

TABLE 46 EETI-II-based binders against LRP6 E1E2 VARIANT LOOP 1 LOOP 2ELISA S/N* LRP6_CKP1 RTNRVKGG GPNGF 3.23 45.49 (SEQ ID NO: 147)(SEQ ID NO: 19) LRP6_CKP2 VNRVRG SGGRD 3.41 41.62 (SEQ ID NO: 148)(SEQ ID NO: 169) LRP6_CKP3 MNHVKARR GPNGF 2.93 40.18 (SEQ ID NO: 149)(SEQ ID NO: 19) LRP6_CKP4 RSVNKI GSSRN 2.82 25.39 (SEQ ID NO: 150)(SEQ ID NO: 170) LRP6_CKP5 VNKIKG GVEGR 3.04 35.71 (SEQ ID NO: 151)(SEQ ID NO: 171) LRP6_CKP6 RNSIKR SVGHG 3.10 37.36 (SEQ ID NO: 152)(SEQ ID NO: 172) LRP6_CKP7 VSNRVNKG GPNGF 3.30 28.96 (SEQ ID NO: 153)(SEQ ID NO: 19) LRP6_CKP8 RGNIIK NESRG 3.23 37.56 (SEQ ID NO: 154)(SEQ ID NO: 173) LRP_CKP9 RSGNTIRKRE GGPGG 2.97 37.62 (SEQ ID NO: 155)(SEQ ID NO: 174) LRP6_CKP10 ASSNSIRQGW GPKSN 3.29 37.38 (SEQ ID NO: 156)(SEQ ID NO: 175) LRP6_CKP11 RSNRIR YGHGD 2.65 36.76 (SEQ ID NO: 157)(SEQ ID NO: 176) LRP_CKP12 RSNKLREARG GSRQD 0.60 6.78 (SEQ ID NO: 158)(SEQ ID NO: 177) LRP6_CKP13 VNSVKR SRGVN 3.28 37.75 (SEQ ID NO: 159)(SEQ ID NO: 178) LRP6_CKP14 GSNKIRPR GPNDF 3.18 43.53 (SEQ ID NO: 160)(SEQ ID NO: 179) LRP6_CKP15 NRIRNS GRGDY 2.03 26.31 (SEQ ID NO: 161)(SEQ ID NO: 180) LRP6_CKP16 SRNSIK ASGSS 3.36 31.11 (SEQ ID NO: 162)(SEQ ID NO: 181) LRP6_CKP17 SNYVKR SPGGR 3.09 35.88 (SEQ ID NO: 163)(SEQ ID NO: 182) LRP6_CKP18 RANRVSGR GPNGF 1.67 18.32 (SEQ ID NO: 164)(SEQ ID NO: 19) LRP6_CKP19 SNRVKVRA GPNGF 3.27 41.96 (SEQ ID NO: 165)(SEQ ID NO: 19) LRP6_CKP20 ENRTKG GFRGT 3.10 38.69 (SEQ ID NO: 166)(SEQ ID NO: 183) LRP6_CKP21 GNKIRA RDRVG 2.80 33.69 (SEQ ID NO: 167)(SEQ ID NO: 184) LRP6_CKP22 ANRVKRTS GPNGF 3.43 42.86 (SEQ ID NO: 168)(SEQ ID NO: 19) *S/N = signal to noise ratio as compared to BSA control

The extracellular domain of the LRP6 consists of four propeller domains(E1-E4) that interact with Frizzled receptors and Wnt proteins topropagate Wnt signaling. Utilizing a modular approach, LRP6distinguishes between Wnt1 or Wnt3a signaling through selective bindingof either its E1-E2 or E3-E4 domains to specific Wnt isoforms,respectively (Hannoush et al. (2010) J. Biol. Chem. 285, 9172-9179). Topharmacologically delineate Wnt1 and Wnt3a signaling arms, we sought toidentify ligands that bind selectively to LRP6 E1-E2.

Of the identified sequences, R1, LRP6 CKP6 and LRP6 CKP19 were generatedin soluble folded form in order to test their pharmacological activityagainst either Wnt1 or Wnt3a signaling. As shown in Tables 47 and 48below, no significant selectivity was observed by R77 towards Wnt1 orWnt3a in a cell-based signaling reporter assay. On the other hand, R1and R19 showed selective inhibition towards Wnt1 signaling relative toWnt3a (160-fold and 11-fold for Wnt1 over Wnt3a, respectively) asmeasured in a luciferase reporter assay, supporting the notion thatthese variants do not target the LRP6 E3-E4 domains (IC₅₀>44 μM).Altogether, the data highlight the specificity of the newly evolvedvariants and their effects in mimicking a motif which occurs in naturalligands. More importantly, the identified variants provide apharmacological means to interrogate Wnt1 and Wnt3 signaling.

TABLE 47 Inhibitory activity of LRP6-binding CKP variants against Wnt1signaling Lrp6-CKP Best Fit IC₅₀ (nM) for 95% confidence (n = 4) Wnt1 (5ng/well) interval (nM) R1 F1 241.7 185.8 to 314.5 R1 F2 193.8 140.1 to268.2 LRP6_CKP6 F1 22,866 13,593 to 38,463 LRP6_CKP6 F2 23,760 14,458 to39,046 LRP6_CKP6 F3 4,625 3,037 to 7,044 LRP6_CKP19 F1 23,132 15,397 to34,754 LRP6_CKP19 F2 49,330 31,391 to 77,520

TABLE 48 Inhibitory activity of LRP6-binding CKP variants against Wnt3asignaling Lrp6-CKP Best Fit IC₅₀ (nM) for 95% confidence (n = 4) Wnt3a(25 ng/well) interval (nM) R1 F1 38,594 16,093 to 92,554  R1 F2 16,5969,037 to 30,478 LRP6_CKP6 F1 350,240     11840 to 1.036 × 10⁷ LRP6_CKP6F2 275,584    24,695 to 3.075 × 10⁶ LRP6_CKP6 F3 Not converge Notconverge LRP6_CKP19 F1 59,287 32,600 to 107,823 LRP6_CKP19 F2 69,82726,179 to 186,252

F1, F2, and F3 in Tables 47 and 48 refer to peak fractions 1, 2, and 3,respectively that were obtained during the purification of R1,LRP6_CKP6, and LRP6_CKP19.

The preceding Examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and fall within the scope of the appendedclaims.

1. A non-naturally occurring cystine knot peptide (CKP) that binds tovascular endothelial growth factor A (VEGF-A), wherein the CKP comprisesthe cystine scaffold structure:Z₁C₁L1C₂L2C₃L3C₄L4C₅L5C₆Z₂ wherein: Z₁ and Z₂ are any amino acid or anunnatural amino acid; L1 is Loop 1 and has a structure selected from thegroup consisting of: X₁X₂X₃X₄X₅X₆ (SEQ ID NO: 2), X₁X₂X₃X₄X₅X₆X₇ (SEQ IDNO: 3), X₁X₂X₃X₄X₅X₆X₇X₈ (SEQ ID NO: 4), X₁X₂X₃X₄X₅X₆X₇X₈X₉ (SEQ ID NO:5), and X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀ (SEQ ID NO: 6), wherein each of X₁-X₁₀ isany amino acid or an unnatural amino acid; L2 is Loop 2 and has thestructure: X₁X₂X₃X₄X₅ (SEQ ID NO: 7), wherein each of X₁-X₅ is any aminoacid or an unnatural amino acid; L3 is Loop 3 and has the structure:X₁X₂X₃ wherein each of X₁-X₃ is any amino acid or an unnatural aminoacid; L4 is Loop 4 and has the structure: X₁, wherein X₁ is any aminoacid or an unnatural amino acid; L5 is Loop 5 and has the structure:X₁X₂X₃X₄X₅ (SEQ ID NO: 7), wherein each of X₁-X₅ is any amino acid or anunnatural amino acid; wherein the unnatural amino acid is selected fromthe group consisting of L-propargylglycine-PEG₆-, L-sulfotyrosine,L-norleucine, L-1-naphthylalanine, L-2-naphthylalanine,L-2-chlorotryptophan, L-3-fluorotyrosine, L-4-fluorophenylalanine,gamma-benzyl-L-proline, gamma-(4-fluoro-benzyl)-L-proline,4-OH-L-proline, 4-fluoro-L-proline,4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine,3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine,3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NHmeta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine,3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine,2-naphthyl-L-alanine, L-4,4′-biphenylalanine,3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine,3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine,4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine,benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine(also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine,cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine(also known as L-tert-Leucine), L-cyclopentylglycine,L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine,3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine,2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine,4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine,L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester ofglycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, andglycine morpholine amide; wherein the CKP binds to VEGF-A with anaffinity of 500 pM or better.
 2. The CKP of claim 1, wherein the CKP hasan altered disulfide bond connectivity—with reference to a wild-typeEcballium elaterium trypsin inhibitor EETI-II protein having the aminoacid sequence set forth in SEQ ID NO: 1; wherein the altered disulfidebond connectivity is C1-C4, C2-C3 and C5-C6.
 3. The CKP of claim 1 orclaim 2, wherein Z₁ and/or Z₂ is G.
 4. A non-naturally occurring cystineknot peptide (CKP) comprising an amino acid selected from the groupconsisting of: GCNIMLPFWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 113),GCNIMLPFWGCGRDFECVYRCICQYYQSCG (SEQ ID NO: 114),GCDVMQPYWGCGPDIDCFVRCLCHWYNSCG (SEQ ID NO: 139),GCDVMQPYWGCGPDIDCLSNCICHWYNSCG (SEQ ID NO: 140),GCNIMLPYWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 142),GCNIXLPFWGCGRDFECMSDCICQYYQSCG (SEQ ID NO: 144), wherein X is norleucine(Nle), GCNIXLPFWGCGRDFECVSQCICQYYQSCG (SEQ ID NO: 145), wherein X isnorleucine (Nle), GCNIXLPYWGCGRDFECMEQCICQYYQSCG (SEQ ID NO: 146),wherein X is norleucine (Nle), and GCDVXQPYWGCGPDIDCLSNCICHWYNSCG (SEQID NO: 224), wherein X is norleucine.
 5. The CKP of claim 4, wherein theCKP binds VEGF-A.
 6. A non-naturally occurring cystine knot peptide(CKP) comprising an amino acid selected from the group consisting of:GCDVX₁QPYWGCGPDI-D/E-CLS-N/K/X₂-CICHWYNSCG (SEQ ID NO: 534),GCDVX₁QPYWGCGPDI-N/K/X₂-CLS-D/E-CICHWYNSCG (SEQ ID NO: 535),GCNIX₁LPYWGCGRDF-D/E-CME-N/K/X₂₋CICQYYQSCG (SEQ ID NO: 538),GCNIX₁LPYWGCGRDF-N/K/X₂-CME-D/E_CICQYYQSCG (SEQ ID NO: 539),GCNIX₁LPFWGCGRDF-D/E-CVS-N/K/X₂-CICQYYQSCG (SEQ ID NO: 540), andGCNIX₁LPFWGCGRDF-N/K/X₂-CVS-D/E-CICQYYQSCG (SEQ ID NO: 541), wherein X₁is norleucine and X₂ is ornithine.
 7. The CKP of claim 6, wherein theCKP binds VEGF-A.
 8. A non-naturally occurring cystine knot peptide(CKP) that binds to VEGF-A, wherein the CKP comprises the cystinescaffold structure:Z₁C₁L1C₂L2C₃L3C₄L4C₅L5C₆Z₂ wherein: Z₁ and Z₂ are any amino acid, morethan one amino acid, or an unnatural amino acid; L1 is Loop 1 and has astructure selected from the group consisting of: X₁X₂X₃X₄X₅X₆X₇X₈,X₁X₂X₃X₄X₅X₆X₇X₈X₉, and X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀, wherein each of X₁-X₁₀ isany amino acid or an unnatural amino acid; L2 is Loop 2 and has thestructure: X₁X₂X₃X₄X₅, wherein each of X₁-X₅ is any amino acid or anunnatural amino acid; L3 is Loop 3 and has the structure: X₁X₂X₃ whereineach of X₁-X₃ is any amino acid or an unnatural amino acid; L4 is Loop 4and has the structure: X₁, wherein X₁ is any amino acid or an unnaturalamino acid; L5 is Loop 5 and has the structure: X₁X₂X₃X₄X₅, wherein eachof X₁-X₅ is any amino acid or an unnatural amino acid; wherein theunnatural amino acid selected from the group consisting ofL-propargylglycine-PEG₆-, L-sulfotyrosine, L-norleucine,L-1-naphthylalanine, L-2-naphthylalanine, L-2-chlorotryptophan,L-3-fluorotyrosine, L-4-fluorophenylalanine, gamma-benzyl-L-proline,gamma-(4-fluoro-benzyl)-L-proline, 4-OH-L-proline, 4-fluoro-L-proline,4-[4-(trifluoromethyl)-benzyl]-L-proline, 3,4-difluoro-L-phenylalanine,3,4-dichloro-L-phenylalanine, 4-chloro-L-phenylalanine,3-F,4-Cl-L-phenylalanine, 2-pyridone(NH para)-L-alanine, pyridone(NHmeta)-L-alanine, 3-(1-N-methyl indole)-L-alanine, 3-(1-N-ethylindole)-L-alanine, 3-(1-N-isopropyl indole)-L-alanine,3-(5-aza-indole)-L-alanine, 4-methyl-L-phenylalanine,2-naphthyl-L-alanine, L-4,4′-biphenylalanine,3-(3-quinolinyl)-L-alanine, 3-(2-quinolinyl)-L-alanine,3-(2-quinoxalinyl)-L-alanine, 4-methyl-2-pyridyl-alanine,4-ethyl-2-pyridyl-L-alanine, benzothiazole-L-alanine,benzothiophene-L-alanine, 3-isoquinolinyl-L-alanine, t-butyl-L-alanine(also known as L-Nepentyl glycine), 3-cyclobutyl-L-alanine,cyclopentyl-L-alanine, 5,5,5-Trifluoro-L-leucine, t-butyl-L-glycine(also known as L-tert-Leucine), L-cyclopentylglycine,L-cyclobutylglycine, 3,4-hydroxy-L-phenylalanine,3,4-fluoro-L-phenylalanine, 3-fluoro,4-OH-L-phenylalanine,2-chloro-L-tyrosine, 2-methyl-L-tyrosine, 2-ethyl-L-tyrosine,4-(naphthalen-1-ol)-L-alanine, D-serine, L-beta-homoserine,L-beta-alanine, N-alpha-methyl glycine, glycine amide, glycine ester ofglycerol, glycine ester of glycol, glycine ester of oxetane-3-yl, andglycine morpholine amide, and wherein the CKP has an altered disulfidebond connectivity with reference to a wild-type Ecballium elateriumtrypsin inhibitor EETI-II protein having the amino acid sequence setforth in SEQ ID NO: 1; wherein the altered disulfide bond connectivityis C1-C4, C2-C3 and C5-C6; and wherein the CKP has a percent alpha helixcontent of at least 20%.
 9. The CKP of any one of claims 1-8, wherein:(a) the C-terminal carboxyl group of the peptide is capped; (b) theN-terminal amine of the peptide (CKP) is capped; or (c) the C-terminalcarboxyl group and the N-terminal amine of the peptide (CKP) is capped.10. A non-naturally occurring CKP that binds to an epitope on VEGF-Acomprising at least one of the amino acid residues selected from thegroup consisting of: V14, V15, F17, D19, Y21, Q22, Y25, I46, K48, N62,D63, L66, M81, 183, K84, P85, H86, G88, Q89, I91, C104, R105, and P106.11. The CKP of any one of claims 1-10 conjugated to a therapeutic agent.12. The CKP of any one of claims 1-10 conjugated to a label.
 13. Anisolated nucleic acid encoding the CKP of any one of claims 1-10.
 14. Anexpression vector encoding the nucleic acid molecule of claim
 13. 15. Acell comprising the expression vector of claim
 14. 16. A method ofproducing the CKP of any one of claims 1-10 comprising culturing thecell of claim 15 and recovering the CKP from the cell culture.
 17. Amethod of producing the CKP of any one of claims 1-10, comprisingchemically synthesizing the CKP.
 18. A composition comprising the CKP ofany one of claims 1-11 and a pharmaceutically acceptable carrier.
 19. Amethod of treating an ocular disease characterized by angiogenesisand/or vascular permeability or leakage in a subject, comprisingadministering an effective amount of the CKP of any one of claims 1-11or the composition of claim 18 to the subject.
 20. The method of claim19, wherein the ocular disease is an intraocular neovascular diseaseselected from the group consisting of proliferative retinopathies,choroidal neovascularization (CNV), age-related macular degeneration(AMD), diabetic and other ischemia-related retinopathies, diabeticmacular edema, pathological myopia, von Hippel-Lindau disease,histoplasmosis of the eye, retinal vein occlusion (RVO), includingCentral Retinal Vein Occlusion (CRVO) and branched retinal veinocclusion (BRVO), corneal neovascularization, retinalneovascularization, and retinopathy of prematurity (ROP).
 21. The methodof any one of claim 19 or 20, wherein the CKP or the composition isadministered to the subject via an implantable device.
 22. The CKP ofany one of claims 1-11, wherein the CKP is formulated for long actingdelivery.
 23. A formulation comprising the CKP of any of claims 1-11 andPLGA.
 24. A non-naturally occurring cystine knot peptide (CKP) thatbinds to human low density lipoprotein receptor-related protein 6(LRP6), wherein the CKP comprises the cystine scaffold structure:Z₁C₁L1C₂L2C₃L3C₄L4C₅L5C₆Z₂; wherein: Z₁ and Z₂ are any amino acid; L1 isLoop 1 and has a structure selected from the group consisting of:X₁X₂X₃X₄X₅X₆, X₁X₂X₃X₄X₅X₆X₇, X₁X₂X₃X₄X₅X₆X₇X₈, X₁X₂X₃X₄X₅X₆X₇X₈X₉, andX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀, wherein each of X₁-X₁₀ is any amino acid; L2 isLoop 2 and has the structure: X₁X₂X₃X₄X₅, wherein each of X₁-X₅ is anyamino acid; L3 is Loop 3 and has the structure: X₁X₂X₃ wherein each ofX₁-X₃ is any amino acid; L4 is Loop 4 and has the structure: X₁, whereinX₁ is any amino acid; and L5 is Loop 5 and has the structure:X₁X₂X₃X₄X₅, wherein each of X₁-X₅ is any amino acid.